MEMCAL    tSCMOOL 


^^<M>^  (ju-iCf^^ 


CHEMICAL   PHYSIOLOGY 


H  IB 
1907 


PEEFACE 

TO 

THE     SIXTH    EDITION 

I  HAVE  again  subjected  the  book  to  a  thorough  revision,  and  the 
changes  which  are  now  introduced  into  the  practical  exercises  are 
those  which  experience  has  shown  to  be  advisable.  In  the  large 
text  it  has  been  necessary  to  rewrite  a  good  many  parts,  mainly  on 
account  of  our  increased  knowledge  of  the  proteins  and  of  the  way 
they  are  utilised  in  the  body.  The  sections  relating  to  blood  coagula- 
tion and  to  respiration  have  been  much  amplified  in  order  to  include 
many  facts  which  are  the  result  of  recent  research. 

In  my  endeavour  to  bring  the  work  abreast  of  advances  in 
science,  and  at  the  same  time  to  keep  it  within  moderate  limits, 
I  have  to  acknowledge  help  and  valuable  suggestions  from  Mr.  J. 
Barcroft,  M.A.  (especially  in  connection  with  Eespiration),  from 
Professor  T.  G.  Brodie,  F.E.S.,  and  from  my  two  colleagues  at  King's 
College,  Dr.  Lyle  and  Dr.  O.  Eosenheim ;  both  of  these  have  been 
of  great  assistance  to  me  in  reading  the  proof-sheets,  and  Dr.  Lyle 
is  again  responsible  for  the  Index. 

W.  D.  Hallibubton. 
King's  College,  1907. 


J  1089 


Digitized  by  the  Internet  Archive 

in  2007  with  funding  from 

Microsoft  Corporation 


http://www.archive.org/details/essentialsofchemOOhallrich 


CONTENTS 


PAGE 

Introduction 1 


ELEMENTARY  COURSE 

Lessox 

I.     The  Elements  contained  in  Physiological  Compounds  ...  9 

II.     The  Carbohydrates .         .         .13 

III.  The  Fats - 22 

IV.  The  Proteins 27 

V.     The  Proteins  (continued) 29 

VI.     Foods 49 

VII.     The  Digestive  Juices — Saliva  and  Gastric  Digestion  ...  62 

VIII.     The  Digestive  Juices  (continued) — Pancreatic  Digestion  and  Bile  78 

IX.     The  Blood  and  Eespiration 101 

X.     Urine 141 

XI.     Urine  (continued) 156 

XII.     Pathological  Urine         .        .         .         .  ^ 166 

Scheme  for  Detecting  Physiological  Proximate  Principles   .        .171 

ADVANCED   COURSE 

Introduction 175 

Lesson 

XIII.  Carbohydrates 176 

XIV.  Action  of  Malt  upon  Starch 179 

XV.     Crystallisation  of  Egg  Albumin 180 

XVI.     Milk 181 

XVII.     The  Proteoses  ......                          ...  182 


VIU  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

Lesson  page 

XVIII.  Digestion 184 

XIX.  HAEMOGLOBIN   AND   ITS   DERIVATIVES 188 

XX.  Serum 192 

XXI.  Coagulation  of  Blood 194 

XXII.  Muscle  and  Nebvous  Tissue 197 

XXIII.  Urea  and  Chlorides  in  Urine 204 

XXIV.  Phosphates  and  Sulphates  in  Urine 207 

XXV.  Uric  Acid  and  Creatinine        ........  210 

XXVI.  The  Pigments  of  the  Urine 213 


APPENDIX 

HiEMACYTOMETERS *          .* 217 

h.ajmoglobinometebb       .        .        .  .        .        .  *      .  .219 

Polarisation  of  Light 222 

polarimeters 226 

The  Spectro-polarimeter '.'.'.        .        .  220 

Eelation  between  Circular  Polarisation  and  Chemical  Constitution       .  230 

Mercurial  Air-pumps 231 

Analysis  of  Gases 234 

Kjeldahl's  Method  of  Estimating  Nitrogen       .        ."       .         .         .        .  235 

Solutions,  Diffusion,  Dialysis,  Osmosis 236 

INDEX 245 


LIST    OF    ILLUSTRATIONS 


FIG-  -  PAGE 

1.  Dextrose  Crystals .  Frey  17 

2.  Inosite  Crystals  .        .         .         .        .        .         .           Frey  18 

3.  Milk-sugar  Crystals              Frey  19 

4.  Section  of  Pea,  showing  Starch  Grains      .         ,         Yeo,  after  Sachs  20 

5.  Fat  Cells Schdfer  23 

6.  Simple  Warm  Bath 29 

7.  Dialyser 37 

8.  Dialyser 37 

9.  Diagram  of  a  Cell Schafer  45 

10.  Milk Yeo  5S 

11.  Colostrum  Corpuscles HeidenJiain  53 

12.  Yeast  Cells Yeo' s  Physiology  63 

13.  Schizomycetes After  Zopf  64 

14.  Bacillus  Anthracis Koch  65 

15.  Alveoli  of  Serous  Gland Langley  69 

16.  Mucous  Cells Langley  69 

17.  Submaxillary  Gland Heidenhain  69 

18.  Fundus  Gland Klein  71 

19.  Pyloric  Gland Ehstein  71 

20.  Fundus  Gland Langley  72 

21.  Alveolus  of  Pancreas Kuhne  and  Lea  80 

22.  Leucine  Crystals KUhne  86 

23.  Tyrosine  Crystals ^rey  86 

24.  H^MATOiDiN  Crystals .         ,          Frey  89 

25.  Cholesterin  Crystals Frey  93 


X  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

FIG.  PAGR 

26.  Villus  of  Eat  Killed  during  Fat  Absorption     .        .        .     Schafer  98 

27.  Mucous  Membrane  of  Frog's  Intestine  during  Fat 

Absorption ScJuifer  99 

28.  Fibrin  Filaments  and  Blood  Platelets       ....     Schafer  103 

29.  Action  of  Reagents  on  Blood  Corpuscles  ....     Schafer  111 

30.  Oxyh.i:moglobin  Crystals Quairi's  Anatomy  112 

31.  H;emin  Crystals     ...                  Preyer  113 

32.  Diagram  of  Spectroscope .        .        .  116 

33.  Figure  of  Spectroscope  and  Accessories     .        .        .      McKendrick  116 

34.  Arrangement  of  Prisms  in  Direct-vision  Spectroscope       GscJieidlen  117 

35.  Stand  for  Direct- vision  Spectroscope 118 

36.  Absorption  Spectra        .  " Rollett  118 

37.  Absorption  Spectra 119 

38.  Loewy's  Aerotonometer 132 

39.  Dissociation  Curves  of  Blood  and  Hemoglobin  .         .        Bohr  133 

40.  Barcroft's  Blood  Gas  Apparatus 135 

41.  Dupre's  Urea  Apparatus Gamgee  142 

42.  XJrinometer McKendrick  144 

43.  Urea  Crystals Fi-ey  144 

44.  Urea  Nitrate  and  Oxalate Frey  146 

45.  Triple  Phosphate  Crystals  .......  Frey  155 

46.  Uric  Acid  Crystals Frey  157 

47.  Acid  Sodium  Urate Frey  163 

48.  Acid  Ammonium  Urate Frey  163 

49.  Envelope  Crystals  of  Calcium  Oxalate       ....  Frey  164 

50.  Cystin  Crystals Frey  164 

51.  Triple  Phosphate  Crystals Bryant's  Surgery  164 

52.  Calcium  Phosphate  Crystals         ....     Bryant's  Surgery  164 

63.    Albuminometeb  of  Esbach 166 

f)4.  Two  Burettes  on  Stand        .        .         .        .  ;      .                 .       Sutton  167 

55.  Hot-air  Oven  with  Gas  Regulator       ....         Gscheidlen  176 

56.  OsAzoNE  Crystals Coloured  x>lcde  to  face  177 

57.  Absorption  Spectra  op  Hemoglobin,  &c 189 

58.  Photographic  Spectrum  of  Hemoglobin  and  Oxyhemoglobin  Gamgee  190 


LIST  OF  ILLUSTRATIONS  XI 

FIG.  PAGB 

59.  Photographic  Spectrum  of  Oxyhjemoglobin  and  Methjemo- 

GLOBiN Oamgee  190 

60.  Centrifugal  Machine 195 

61.  Absorption  Spectra  of  Mtoh-ematin 199 

62.  A  Desiccator Gscheidlen  199 

63.  Absorption  Spectra  of  Urinary  Pigments   .        .        .  After  Hopkins  215 

64.  Gowers'  Hemacytometer 217 

65.  Oliver's  H.ismacytometer 218 

66.  Gowers'  Hemoglobinometer 219 

67.  Von  Fleischl's  Hemometer 220 

68.  Oliver's  Hjemoglobinometer 221 

69.  Model  to  Illustrate  Polarised  Light 223 

70.  Model  to  Illustrate  Polarised  Light 223 

71.  Model  to  Illustrate  Polarised  Light 224 

72.  Diagram  to  Explain  Polarisation  of  Light 225 

73.  Diagram  to  Explain  Polarisation  of  Light 226 

74.  Soleil's  Saccharimeter 227 

75.  Diagram  of  Optical  Arrangements  in  Soleil's  Saccharimeter           .  227 

76.  Laurent's  Polarimeter 228 

77.  Spectro-polarimeter  of  von  Fleischl 229 

78.  Diagram  of  Asymmetric  Carbon  Atoms 231 

79.  Diagram  of  Pfluger's  Pump 232 

80.  L.  Hill's  Air-pump 233 

81.  Waller's  Apparatus  for  Gas  Analysis         ....       Wall^  234 

82.  Kjeldahl's  Method,  Distilling  Apparatus 235 

83.  Diagram  to  Illustrate  Osmosis 239 


ESSENTIALS 

OF 

CHEMICAL    PHYSIOLOGY 

INTEODUCTION 

Chemical  Physiology  or  Physiological  Chemistry  deals  with  the 

chemical  composition  of  the  body  and  with  the  chemical  changes  it 
undergoes ;  it  also  deals  with  the  composition  of  the  food  which 
enters,  and  the  excretions  which  leave,  the  body. 

When  a  chemist  examines  living  things  he  is  placed  at  a  dis- 
advantage when  compared  with  an  anatomist;  for  the  latter  can 
with  the  microscope  examine  cells,  organisms,  and  structures  in  the 
living  condition.  The  chemist,  on  the  other  hand,  cannot  at  present 
state  anything  positive  about  the  chemical  structure  of  living  matter, 
because  the  reagents  he  uses  will  destroy  the  life  of  the  tissue  he 
is  examining.  There  is,  however,  no  such  disadvantage  when  he 
examines  non-living  matter,  like  food  and  urine,  and  it  is  therefore 
in  the  analysis  of  such  substances  that  chemical  physiology  has 
made  very  important  advances,  and  the  knowledge  so  obtained  is 
of  the  greatest  practical  interest  to  the  student  and  practitioner  of 
medicine. 

The  animal  organism  is  in  its  earliest  embryonic  state  a  single 
cell;  as  development  progresses  it  becomes  an  adherent  mass  of 
simple  cells.  In  the  later  stages  various  tissues  become  differen- 
tiated from  each  other  by  the  cells  becoming  grouped  in  different 
ways  by  alteration  in  the  shape  of  the  cells,  by  deposition  of  inter- 
cellular matter  between  the  cells,  and  by  chemical  changes  in  the 
living  matter  of  the  cells  themselves.  Thus  in  some  situations  the 
cells  are  grouped  into  the  various  epithehal  linings;  in  others  the 
n  B 


2  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

cells  become  elongated,  and  form  muscular  fibres  ;  in  the  connective 
tissues  we  have  a  preponderating  amount  of  intercellular  material, 
which  may  become  permeated  with  fibres,  or  be  the  seat  of  the  deposi- 
tion of  calcareous  salts,  as  in  bone.  Instances  of  chemical  changes 
in  the  cells  themselves  are  seen  on  the  surface  of  the  body,  where  the 
superficial  layers  of  the  epidermis  become  horny  {i.e.  filled  with  the 
chemical  substance  called  keratin) ;  in  the  mucous  salivary  glands, 
where  the  cells  become  filled  with  mucin,  which  they  subsequently 
extrude;  and  in  adipose  tissue,  where  they  become  filled  with 
fat. 

In  spite  of  these  changes,  the  variety  of  which  produces  the  great 
complexity  of  the  adult  organism,  there  are  many  cells  which  still 
retain  their  primitive  structure :  notable  among  these  are  the  white 
corpuscles  of  the  blood. 

A  cell  may  be  defined  as  a  mass  of  living  material  containing  in 
its  interior  a  more  solid  structure  called  the  nucleus.  The  nucleus 
exercises  a  controlling  influence  over  the  nutrition  and  subdivision 
of  the  cell. 

The  living  substance  is  usually  pervaded  with  granules  :  one  of 
these  minute  particles  called  the  centrosome  exercises  an  attractive 
influence  on  the  granules  and  fibrils  of  the  protoplasm  in  its  neighbour- 
hood, and  the  appearance  so  produced  is  called  the  attraction  sphere. 
The  attraction  sphere  becomes  specially  prominent,  and  divides  into 
two  when  the  cell  is  about  to  divide ;  this  usually  precedes  the 
division  of  the  nucleus. 

Living  material  is  called  protoplasm,  and  protoplasm  is  charac- 
terised by  (1)  irritability — that  is,  the  property  of  responding  by  some 
change  when  subjected  to  the  influence  of  an  external  agent  or 
stimulus  :  the  most  obvious  of  these  changes  is  movement  (amoeboid 
movement,  ciliary  movement,  muscular  movement,  &c.);  (2)  its  power 
of  assimilation — that  is,  it  is  able  to  convert  into  protoplasm  the 
nutrient  material  or  food  which  is  ingested  ;  (3)  its  power  of  growth 
— this  is  a  natural  consequence  of  its  power  of  assimilation ;  (4)  its 
power  of  reproduction — this  is  a  variety  of  growth ;  and  (5)  its  power 
to  excrete,  to  give  out  waste  materials,  the  products  of  its  other 
activities. 

Of  all  the  signs  of  life,  those  numbered  2  and  5  in  the  foregoing 
list  are  the  most  essential.  Living  material  is  in  a  continual  state  of 
unstable  chemical  equilibrium,  building  itself  up  on  the  one  hand, 
breaking  down  on  the  other ;  the  term  used  for  the  sum  total  of  these 
intra-molecular  rearrangements  is  metabolism.  The  chemical  sub- 
stances in  the  protoplasm  which  are  the  most  important  from  this 


INTRODUCTION  3 

point  of  view  are  the  complex  nitrogenous  compounds  called  Proteins,^ 
So  far  as  is  at  present  known,  protein  material  is  never  absent  from 
living  substance,  and  is  never  present  in  anything  else  thaii  that 
which  is  alive  or  has  been  formed  by  the  agency  of  living  cells.  It 
may  therefore  be  stated  that  Protein  Metabolism  is  the  most  essential 
characteristic  of  vitality. 

The  chemical  structure  of  protoplasm  can  only  be  investigated 
after  the  protoplasm  has  been  killed.  The  substances  it  yields  are 
(1)  Water  ;  protoplasm  is  semi-iluid,  and  at  least  three-quarters  of 
its  weight,  often  more,  are  due  to  water.  (2)  Proteins.  These  are 
the  most  constant  and  abundant  of  the  solids.  A  protein  or  albu- 
minous substance  consists  of  carbon,  hydrogen,  nitrogen,  oxygen, 
with  sulphur  and  phosphorus  in  small  quantities  only.  In  nuclein, 
a  protein-like  substance  obtained  from  the  nuclei  of  cells,  phosphorus 
is  more  abundant.  The  protein  obtained  in  greatest  abundance  from 
the  cell-protoplasm  is  nucleo -protein  :  that  is,  a  compound  of  protein 
with  varying  amounts  of  nucleiji.  White  of  egg  is  a  familiar  instance 
of  an  albuminous  substance  or  protein,  and  the  fact  (which  is  also 
familiar)  that  this  sets  into  a  solid  on  boiling  will  serve  as  a  reminder 
that  the  greater  number  of  the  proteins  found  in  nature  have  a 
similar  tendency  to  coagulate  under  the  influence  of  heat  and  other 
agencies.  (3)  Various  other  substances  occur  in  smaller  proportions, 
the  most  constant  of  which  are  lecithin,  a  phosphorised  fat ;  chole- 
sterin,  a  monatomic  alcohol :  and  inorganic  salts,  especially  phos- 
phaucs  and  chlorides  of  calcium,  sodium,  and  potassium. 

It  will  be  seen  from  this  rapid  survey  of  the  composition  of  the 
body  how  many  are  the  substances  which  it  is  necessary  we  should 
study  ;  the  food  from  which  it  is  built  up  is  also  complex,  for  animals 
do  not  possess,  to  such  an  extent  as  plants  do,  the  power  of  building 
up  complex  from  simple  materials. 

The  substances  out  of  which  the  body  is  built  consist  of 
chemical  elements  and  of  chemical  compounds,  or  unions  of  these 
elements. 

The  elements  found  in  the  body  are  carbon,  hydrogen,  nitrogen, 
oxygen,  sulphur,  phosphorus,  fluorine,  chlorine,  iodine,  silicon, 
sodium,  potassium,  calcium,  magnesium,  lithium,  iron,  and  occa- 
sionally manganese,  copper,  and  lead. 

Of  these  very  few  occur  in  the  free  state.  Oxygen  and  nitrogen  (to 
a  small  extent)  are  found  dissolved  in  the  blood-plasma ;  hydrogen  is 

'  In  most  English  text-books  these  substances  have  hitherto  been  called 
Proteids.  The  change  to  Protein  brings  English,  American,  and  German  usage 
into  harmony. 

b2 


4  ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 

formed  by  putrefaction  in  the  alimentary  canaL  With  some  few  ex- 
ceptions such  as  these,  the  elements  enumerated  above  are  found 
combined  with  one  another  to  form  compounds. 

The  compounds,  or,  as  they  are  often  termed  in  physiology,  the 
proximate  prhiciples,  found  in  the  body  are  divided  into — 

(1)  Mineral  or  inorganic  compounds. 

(2)  Organic  compounds,  or  compounds  of  carbon. 

A  convenient  practical  method  of  grouping  these  proximate 
principles  of  the  body  and  of  food  is  the  following  : — 

(  Water. 

Inorganic    .         .         .         .       j  Salts — e.,<7.chlorides  and  phosphates  of  sodium 

(  and  calcium. 

/                                  j  Proteins — e.g.  albumin,  myosin,  gelatin. 

Nitrogenous         \  Simpler   nitrogenous  bodies — e.g.   lecithin, 

i  creatine,  urea. 

Organic    ■<                                   (  Fats — e.g.  butter,  fats  of  adipose  tissue. 

^,         .,                   J  Carbohydrates — e.g  sugar,  starch. 

JNon-mtrogenous  j  simple  organic  bodies— e.g.  alcohol,  chole- 

V                                  V  sterin,  vegetable  acids  and  salts,  lactic  acid. 

Many  of  the  substances  enumerated  above  only  occur  in  small 
quantities.  The  most  important  are  the  inorganic  substances,  water 
and  salts ;  and  the  organic  substances,  proteins,  carbohydrates,  and 
fats.  It  is  necessary  in  our  subsequent  study  of  the  principles  of 
chemical  physiology  that  we  should  always  keep  in  mind  this  simple 
classification  ;  the  subdivision  of  organic  substances  into  proteins, 
fats,  and  carbohydrates  forms  the  starting  point,  the  A  B  C,  as  one 
might  say,  of  chemical  physiology. 

I  shall  conclude  this  introductory  chapter  by  giving  a  list  of  the 
apparatus  and  reagents  necessary  for  a  practical  study  of  the  subject, 
and  some  tables  to  which  it  will  be  often  found  convenient  to  refer. 

The  following  set  of  reagents  conveniently  contained  in  4  to  6  oz.  glass 
stoppered  bottles  should  be  provided  for  each  two  students  : — 

Sulphuric  acid,  concentrated. 
„  „      25  per  cent. 

„  „      01  per  cent. 

Nitric  acid,  concentrated. 
Fuming  nitric  acid. 
Hydrochloric  acid,  concentrated. 
„  „     0-2  per  cent.^ 

Acetic  acid,  glacial. 
„        „      20  per  cent. 

?>         "      .  ^        " 
Glyoxylic  acid. 
Formaldehyde. 

'  Made  by  adding  994  c.c.  of  water  to  C  c.c.  of  the  concentrated  hydrochloric 
acid  of  the  British  Pharmacopoeia. 


INTRODUCTION  5 

Caustic  potash,  20  per  cent. 

»       0-1 
Ammonia. 

Sodimn  carbonate,  1  per  cent. 
Ammonium  sulphide  solution. 
Ammonium  sulphate,  saturated  solution. 
Silver  nitrate,  1  per  cent. 
Barium  chloride,  saturated  solution. 
Ammonium  molybdate  solution. 
Millon's  reagent  ^  • 

Solution  of  ferrocyanide  of  potassium. 

„  litmus. 

„  sodium  phosphate. 

„  iodine  in  potassium  iodide. 

„  ferrous  sulphate. 

„  ferric  chloride. 

„  sodium  nitroprusside. 

Alcohol. 
Ether. 

Esbach's  reagent.^ 

Solution  of  copper  sulphate,  1  per  cent. 
Fehling's  solution. 
Lime  water. 

The  following  additional  reagents  will  be  required  by  those  taking  the 
advanced  course : — 

Solution  of  mercuric  chloride. 

„  potassium  ferricyanide. 

Sodium  carbonate,  saturated  solution. 

„       chloride,  saturated  solution. 

„  „         10-per-cent.  solution. 

Magnesium  sulphate,  saturated  solution. 
Baryta  mixture.^ 
Sodium  acetate  solution.'* 
Phosphoric  acid,  0'5  per  cent. 

In  addition  to  these,  there  should  be  kept  in  stock  in  the  laboratory,  to 
be  given  out  for  the  lessons  in  which  they  are  used,  the  following :  — 

Solid  sodium  chloride. 

„      magnesium  sulphate. 

„     ammonium  sulphate- 

„      sodio-magnesium  sulphate. 
Standard   solution   of   uranium   acetate   or   nitrate   for   estimating 
phosphates.^ 

'  Mercury  is  dissolved  in  its  own  weight  of  strong  nitric  acid.  The  solution  so 
obtained  is  diluted  with  twice  its  volume  of  water.  The  decanted  clear  liquid  is 
Millon's  reagent. 

2  Ten  grammes  of  picric  acid  and  20  grammes  of  citric  acid  are  dissolved  in 
800  to  900  c.c.  of  boiling  water,  and  then  sufficient  water  added  to  make  up  a  litre. 

^  Made  by  mixing  1  volume  of  barium-nitrate  solution  with  2  of  barium- 
hydrate  solution,  both  saturated  in  the  cold. 

*  Prepared  as  follows : — Sodium  acetate,  100  grammes  ;  water,  900  c.c. ;  glacial 
acetic  acid,  100  c.c. 

^  Instructions  how  to  make  standard  solutions  will  be  given  in  the  lessons 
where  they  are  used. 


6  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

Standard  solution  of  mercuric  nitrate  for  estimating  urea. 

„  „  silver         „  „  chlorides. 

Caustic  soda,  40  per  cent. 
Bromine. 

Solution  of  potassium  bichromate. 
Phenyl  hydrazine  hydrochloride. 
Solid  sodium  acetate. 
Phospho-tungstic  acid. 
Glacial  phosphoric  acid. 
Dry  cupric  oxide. 
Soda-Ume. 

Each  student  should  be  provided  with — 

A  Bunsen  burner. 

1  dozen  test-tubes  in  test-tube  stand. 

2  or  3  4-oz.  flasks. 

2  flat  porcelain  dishes. 

2  or  4  4-oz.  beakers. 

2  small  glass  funnels  and  a  funnel  stand. 

A  glass  stirring  rod  and  a  small  pipette. 

1  burette. 

An  iron  tripod  with  wire  gauze. 

Filter  papers  and  litmus  papers. 

A  100-c.c.  cylindrical  measuring  glass. 

A  thermometer  marked  in  degrees  Centigrade. 

A  urinometer. 

A  tin  can  on  a  stand  to  be  used  as  a  water-bath. 

Apparatus  which  is  not  so  frequently  used,  such  as  that  employed  in 
generating  carbonic  anhydride,  carbonic  oxide,  or  sulphuretted  hydrogen, 
may  be  given  out  as  required.  The  laboratory  should  also  possess  a  good 
balance,  with  its  accessories,  water  and  air  baths,  kept  at  various  temperatures, 
retorts,  and  analytical  apparatus  generally.  The  microscope,  polarimeter, 
spectroscope,  dialyser,  are  also  frequently  employed  in  chemico-physiological 
investigations.  Apparatus  and  reagents  for  carrying  out  the  Kjeldahl 
process  are  also  necessary. 


WEIGHTS  AND   MEASUKES 

The  weights  and  measures  usually  employed  in  science  are  those  of  the 
metric  system ;  but  as  in  this  country  the  practical  physician  still  largely 
uses  English  grains  and  ounces,  we  may  compare  the  two  systems  in  the 
following  way : — 

Weights 

(English  System) 

1  grain  =     0*0648  gramme 

1  ounce  =  437'5  grains  =   28*3595  grammes 

1  lb.  =  16  oz.  =  7,000  grains  =  453*5925 

The  scruple  =  20  grains  =  1*296  gramme,  and  the  drachm  =  60  grains 
=  3*888  grammes,  are  retained  in  use,  but  neither  is  an  aliquot  part  of 
the  ounce  ;  though  for  practical  purposes  an  ounce  is  considered  to  consist  of 
8  drachms. 


1  milligramme  - 
1  centigramme  = 
1  decigramme  = 
1  gramme 
1  decagramme  =  10 
1  hectogramme  =  100 
1  kilogramme  =  1,000 


INTEODUCTION 

(Metric  System) 

0*001  gramme 
0-01         „ 
0-1 


grammes 


0-015432  grain 
0-154323     „ 
1-543235     ,; 
15-43235  grains 
154-3235 
1543-235 
=      15432-35 
=   2  lb.  3  oz.  119-8  „ 


Measures  of  Length 

(English  System) 

1  inch  =  25-4  millimetres 

1  foot  =  12  inches  =  304-8  millimetres 

(Metric  System) 

The  standard  of  length  is  the  metre  ;  subdivisions  and  multiples  of  which 
with  the  prefixes  milli-,  centi-,  and  deci-,  on  the  one  hand,  and  deca-,  hecto- 
and  kilo-,  on  the  other,  have  the  same  relation  to  the  metre  as  the  subdivi- 
sions and  multiples  of  the  gramme,  in  the  table  just  given,  have  to  the 
gramme,  thus  : 

1  millimetre    =  0-001  metre 

1  centimetre    =  0-01        „ 

1  decimetre     =  0-1  „ 

1  metre 


0-03937  inch 
0-3937       „ 
3-93707  inches 
39-37079     „ 


1  minim 

1  fluid  drachm  =  60  minims 

1  fluid  ounce  =  8  fluid  drachms 

1  pint  =  20  fluid  ounces 

1  gallon  =  8  pints 


Measures  of  Capacity 
(English  System) 

=       0*59  cubic  centimetre 
3-549  cubic  centimetres 
28-398 
567-936 

4-54837  litres 


(Metric  System) 

In  the  metric  system  the  measures  of  capacity  are  intimately  connected 
with  the  measures  of  length ;  we  thus  have  cubic  millimetres,  cubic  centi- 
metres, and  so  forth.  The  standard  of  capacity  is  the  litre,  which  is  equal  to 
1,000  cubic  centimetres  ;  and  each  cubic  centimetre  is  the  volume  of  1  gramme 
of  distilled  water  at  4°  C.i 

1  cubic  centimetre  (generally  written  c.c.)  =  16-931  minims. 

1  litre  =  1,000  c.c.  -  1  pint  15  oz.  2  drs.  11  m.  -  35-2154  fluid  ounces. 

1  cubic  inch  =  16-365  c.c. 


THEKMOMETEIC   SCALES 

The  scale  most  frequently  used  in  this  country  is  the  Fahrenheit  scale  ; 
in  this  the  freezing-point  of  water  is  32°,  and  the  boiling  point  212°.  On  the 
Continent  the  Reaumur  scale  is  largely  employed,  in  which  the  freezing- 
point  is  0°,  and  the  boiling-point  80°.     In  scientific  work  the  Centigrade 

'  4°  C.  is  the  temperature  at  which  water  has  the  greatest  density.  For  prac- 
tical purposes  measures  are  more  often  constructed  so  that  a  cubic  centimetre  holds 
a  gramme  of  water  at  16°  C,  which  is  about  the  average  temperature  of  rooms. 
The  true  cubic  centimetre  contains  only  0-999  gramme  at  16°  C 


8 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


scale  has  almost  completely  taken  the  place  of  these ;  in  this  system  the 
freezing-point  is  0°  and  the  boiUng-point  100°. 

To  convert  degrees  Fahrenheit  into  degrees  Centigrade,  subtract  32  and 
multiply  by  f ,  or  C  =  (F  —  32)  f .  Conversely,  degrees  Centigrade  may  be 
converted  into  degrees  Fahrenheit  by  the  following  formula  :  F  =  §C  +  32. 


TENSION   OF  AQUEOUS  VAPOUE  IN    MILLIMETRES   OF 
MERCURY   FROM    10^   TO   25°  C. 


10°.  9-126 
11°.  9-751 
12°.  10-421 
13^  11-130 


14°.  11-882 

15°.  12-677 

16°.  13-519 

17".  14-409 


18°.  15-351 
19^  16-345 
20°.  17-396 
21°.  18-505 


22°.  19-675 
23^  20-909 
24°.  22-211 
25°.  23-582 


TABLE   OF   THE   DENSITY  OF  WATER  AT   TEMPERATURES 
BETWEEN   0°  AND  30°   C. 


0°.  0-99988 
1°.  0-99993 
2°.  0-99997 
3°.  0-99999 
4°.  1-00000 
6°.  0-99999 
6°.  0-99997 
7°.  0-99994 


8°-  0-99988 
9°.  0-99982 
10°.  0-99974 
11°.  0-99965 
12°.  0-99955 
13°.  0-99942 
14°.  0-99930 
15°.  0-99915 


16°.  0-99900 
17°.  0-99884 
18°.  0-99866 
19°.  0-99847 
20°.  0-99827 
21°.  0-99806 
22°.  0-99785 
23°.  0-99762 


24°.  0-99738 
25°.  0-99714 
26°.  0-99689 
27  \  0-99662 
28°.  0-99635 
29^  0-99607 
30°.  0-99579 


SYMBOLS  AND  ATOMIC  WEIGHTS   OF  THE   PRINCIPAL 
ELEMENTS  ' 


Aluminiun 

1   Al 

27-1 

Fluorine 

F 

19-0 

Oxygen 

0 

16-0 

Antimony 

Sb  120-2 

Gold 

Au 

197-2 

Phosphorus 

i  P 

31-0 

Arsenic 

As 

75-0 

Hydrogen 

H 

1-0 

Platinum 

Pt 

194-8 

Barium 

Ba 

137-4 

Iodine 

I 

126-97 

Potassium 

K 

39-15 

Bismuth 

Bi 

208-5 

Iron 

Fe 

55-9 

Silver 

Ag 

107-93 

Boron 

B 

11-0 

Lead 

Pb 

206-9 

Silicon 

Si 

28-4 

Bromine 

Br 

79-96 

Magnesium 

Mg 

24-36 

Sodium 

Na 

23-05 

Cadmium 

Cd 

112-4 

Manganese 

Mn 

55-0 

Strontium 

Sr 

87-6 

Calcium 

Ca 

40-1 

Mercury 

Hg 

200-0 

Sulphur 

S 

32-06 

Carbon 

C 

12-0 

Nickel 

Ni 

58-7 

Tin 

Sn 

119-0 

Chlorine 

CI 

35-45 

Nitrogen 

N 

1404 

Tungsten 

W 

184-0 

Copper 

Cu 

63-6 

Osmium 

Os 

191-0 

Zinc 

Zn 

65-4 

'  The   above 

atomic 

weights   are 

taken 

on   the 

basis  that  0  =  16; 

that  of 

hydrogen  will  then  be  1-008. 


ELEMENTAEY    COUESE 

LESSON  I 
THE  ELEMENTS  CONTAINED  IN  PHYSIOLOGICAL   COMPOUNDS 

1.  Take  a  fragment  of  meat  about  the  size  of  a  pea  and  place  it  in  a  porcelain 
crucible  over  a  Bunsen  flame.  Note  that  it  chars,  showing  the  presence  of 
carbon,  and  that  it  gives  off  the  unpleasant  odour  of  burning  flesh,  which  is 
due  to  the  fact  that  it  contains  the  nitrogenous  substances  called  proteins.  In 
course  of  time  the  organic  material  is  completely  burnt  up,  and  a  small 
amount  of  white  ash  or  inorganic  material  is  left  behind. 

2.  Kepeat  the  experiment  with  a  pure  organic  substance  like  sugar. 
Note  that  no  ash  is  left.  Charring,  as  before,  indicates  the  presence  of  carbon, 
but  there  is  no  characteristic  smell  of  burning  nitrogenous  substances  (absence 
of  nitrogen). 

_  3.  The  tests  for  carbon  depend  on  the  fact  that  when  this  element  is 
oxidised  it  gives  rise  to  carbon  dioxide  ;  the  test  for  hydrogen  depends  on 
the  fact  that  when  this  element  is  oxidised  it  gives  rise  to  water.  If  all  the 
carbon  dioxide  and  water  formed  by  oxidation  from  a  weighed  amount  of 
any  organic  substance  under  examination  are  collected  and  estimated,  the 
amount  of  carbon  and  hydrogen  respectively  which  it  contains  can  be  easily 
calculated.  The  following  exercises,  however,  deal  only  with  the  qualitative 
detection  of  these  elements. 

4.  Tests  for  Carbon. — The  following  tests  can  be  carried  out  with  sugar. 
(a)  When  burnt  in  the  air  it  chars  and  subsequently  the  carbon  entirely 

disappears,  passing  off  in  combination  with  oxygen  as  carbon  dioxide  (carbonic 
acid  gas). 

(h)  Mix  some  of  the  powdered  sugar  in  a  dry  mortar  with  about  ten  times 
the  quantity  of  cupric  oxide  (which  has  been  freed  from  water  by  previous 
heating) ;  place  the  mixture  in  a  dry  test-tube  provided  with  a  rubber  cork 
perforated  by  a  bent  glass  tube  which  dips  into  either  lime  water  or  baryta 
water.  Heat  the  tube  over  a  Bunsen  flame,  and  as  the  carbon  of  the  sugar 
becomes  oxidised  carbon  dioxide  comes  off  and  causes  a  white  precipitate  of 
calcium  or  barium  carbonate,  as  the  case  may  be. 

5.  Test  for  Hydrogen. — In  the  experiment  just  described  (4  h)  note  that 
drops  of  water  due  to  oxidation  of  hydrogen  condense  in  the  colder  parts  of 
the  test-tube. 

6.  Tests  for  Nitrogen. — The  greater  number  of  tests  for  this  element  are 
due  to  the  circumstance  that  on  the  breaking  up  of  organic  substances  which 
contain  it,  it  is  given  off  as  ammonia.  If  the  ammonia  is  all  collected  and 
estimated,  the  amount  of  nitrogen  can  be  easily  calculated.  Kjeldahl's 
method  for  carrying  out  this  quantitative  analysis  is  described  in  the  Appendix. 
The  following  exercises,  however,  are  qualitative  only. 


10  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

(a)  The  characteristic  odour  of  burning  flesh,  horn,  hair,  feathers,  &c.,  has 
been  already  noted,  and,  though  only  a  rough  test,  is  very  trustworthy. 

(6)  Take  a  little  dried  albumin  and  mix  it  thoroughly  in  a  mortar  with 
about  twenty  times  the  amount  of  soda-lime  and  heat  in  a  test-tube  over  a 
Bunsen  flame.  Ammonia  comes  off  in  the  vapours  produced,  and  may  be 
recognised  by  (i.)  its  odour ;  (ii.)  it  turns  moistened  red  litmus  paper  (held  over 
the  mouth  of  the  tube)  blue  ;  (iii.)  it  gives  off  white  fumes  with  a  glass  rod 
(held  over  the  mouth  of  the  tube)  which  has  been  dipped  in  hydrochloric  acid. 

(c)  Mix  some  dried  albumin  with  about  ten  times  its  weight  of  a  mixture 
of  equal  parts  of  magnesium  powder  and  anhydrous  sodium  carbonate.  A 
small  quantity  of  the  mixture — such  as  would  lie  on  the  end  of  a  penknife — 
is  then  carefully  heated  in  a  dry  test-tube  and  finally  heated  more  strongly 
for  about  half  a  minute  to  red  heat.  Dip  the  tube  while  still  glowing  into 
a  beaker  containing  a  few  c.c.  of  distilled  water ;  the  tube  will  break  and  its 
contents  mix  with  the  water.  Filter  and  label  the  filtrate  A ;  add  to  this 
filtrate  a  little  strong  solution  of  potash,  one  or  two  drops  of  cold  saturated 
solution  of  ferrous  sulphate  and  a  drop  of  ferric  chloride  solution.  Bring 
the  mixture  to  boiling  point,  then  cool  and  acidify  with  hydrochloric  acid. 
The  fluid  becomes  bluish  green,  and  gradually  a  precipitate  of  Prussian  blue 
separates  out.  This  test  is  due  to  the  fact  that  some  of  the  nitrogen  is 
fixed  as  sodium  cyanide,  and  this  gives  the  Prussian  blue  reaction  with  the 
reagents  added. 

7.  Tests  for  SuljjJmr. — (a)  In  the  foregoing  test  (6  c)  the  sulphur  of  the 
albumin  combines  with  the  sodium  to  form  sodium  sulphide.  This  may  be 
detected  by  taking  some  of  the  filtrate  A  and  adding  freshly  prepared  solution 
of  sodium  nitro-prusside  ;  a  reddish  violet  colour  forms. 

(6)  Test  for  loosely  combined  Sulphur. — Add  two  drops  of  a  neutral 
lead  acetate  solution  to  a  few  c.c.  of  caustic  soda  solution.  The  precipitate 
of  lead  hydroxide  which  is  first  formed  soon  dissolves.  Heat  a  small  portion 
of  the  albumin  with  this  alkaline  solution.  The  mixture  turns  black  in  con- 
sequence of  the  formation  of  lead  sulphide,  part  of  the  sulphur  present  in 
albumin  in  the  unoxidised  form  having  been  split  oft"  from  it  by  the  caustic 
soda  as  sodium  sulphide. 

(c)  Take  some  dried  albumin  and  fase  with  a  mixture  of  potash  and 
potassium  nitrate.  Cool ;  dissolve  in  water  and  filter.  The  filtrate  will  give 
the  following  tests  for  sulphates : — Acidulate  with  hydrochloric  acid  and  add 
barium  chloride ;  a  white  precipitate  of  barium  sulphate  is  produced. 

{d)  Take  some  solution  of  albumin  and  heat  in  an  open  dish  in  a  fume 
cupboard  for  at  least  an  hour  with  large  excess  of  fuming  nitric  acid,  renewing 
the  acid  from  time  to  time  as  necessary.  The  resulting  fluid  will  give  the 
test  for  sulphate  as  in  c. 

8.  Test  for  Phospliorus. — The  two  tests  just  described  (7  c  and  d)  maybe 
repeated  with  some  substance  (such  as  caseinogen,  nucleoprotein,  or  lecithin) 
which  contains  phosphorus  in  organic  combination ;  or  the  organic  matter 
may  be  more  conveniently  destroyed  by  Neumann's  method,  which  consists 
in  heating  it  with  a  mixture  of  sulphuric  and  nitric  acids.  The  resulting 
fluid  in  each  case  gives  the  following  test  for  phosphates  : — Mix  it  with  half 
its  volume  of  nitric  acid  ;  add  ammonium  molybdate  in  excess  and  boil ;  a 
yellow  crystalline  precipitate  falls. 

The  reactions  described  in  the  foregoing  exercises  show  how  the 
processes  of  pure  chemistry  may  be  employed  for  the  detection  of 
some  of  the  most  important  elements  that  occur  in  substances  of 
physiological  importance,  and  thus  form  a  fitting  introduction  to  a 
study  of  physiological  chemistry. 


ELEMENTS   CONTAINED  IN   PHYSIOLOGICAL  COMPOUNDS      H 

They  show,  in  the  first  instance,  how  the  substances  with  which 
we  have  to  deal  fall  under  the  two  main  categories  of  organic  and 
inorganic.  In  some  of  the  tissues  of  the  body,  like  bone  and  tooth, 
the  inorganic  or  mineral  material  is  in  excess,  but  in  the  softer 
portions  of  the  organism  the  organic  compounds  are  in  great  pre- 
ponderance. 

Organic  chemistry  is  sometimes  defined  as  the  chemistry  of  the 
carbon  compounds  ;  carbon  is  in  all  cases  present,  and  is  usually  the 
most  abundant  element. 

The  most  important  of  the  nitrogenous  substances  are  the 
proteins,  as  already  explained  in  the  introductory  chapter,  and  the 
detection  and  estimation  of  nitrogen  are  thus  exercises  of  the  highest 
interest. 

All  the  proteins  contain  a  small  amount  of  sulphur ;  keratin,  or 
horny  material,  contains  more  than  most  of  them  do. 

Phosphorus  is  another  element  of  considerable  importance,  being 
present  in  nuclein  and  nucleo-proteins,  and  also  in  certain  complex 
fats,  of  which  lecithin  may  be  taken  as  a  type.  Iodine  occurs  united 
to  protein  material  in  the  colloid  substance  of  the  thyroid  gland  ;  iron 
in  the  pigment  of  the  blood  called  haemoglobin ;  sodium,  calcium, 
potassium,  and  other  metals  in  the  inorganic  substances  of  the  body. 
It  would,  however,  lead  us  too  far  into  the  regions  of  pure  chemistry 
to  undertake  exercises  for  the  detection  of  these  and  other  elements 
which  might  be  mentioned,  and  have  been  already  commented  upon. 
The  teacher  of  physiological  chemistry  is  bound  to  assume  that  the 
students  who  come  before  him  have  already  passed  through  a  course 
of  ordinary  chemistry. 

The  main  interest  of  the  exercises  selected  as  types  lies  in  their 
physiological  application.  As  a  rule  an  element  is  detected  by 
breaking  up  or  oxidising  the  more  or  less  complex  molecule  in  which 
it  occurs  into  substances  of  simpler  nature,  and  then  performing  tests 
for  these  simpler  products.  Thus  carbon  is  identified  by  the  forma- 
tion of  carbon  dioxide,  nitrogen  by  the  formation  of  ammonia,  and  so 
forth. 

A  great  many  reactions  which  can  be  performed  in  the  test-tube 
imitate  those  which  are  performed  in  the  body.  Eeactions  in  vitro 
and  in  vivo,  to  use  the  technical  phrases,  often,  though  not  always, 
run  parallel.  Life,  from  one  point  of  view,  is  a  process  of  com- 
bustion or  oxidation  ;  the  fuel  is  supplied  by  the  food ;  this  becomes 
assimilated,  and  so  forms  an  integral  part  of  the  living  substance  of 
the  body  ;  it  is  then  burnt  up  by  the  oxygen  brought  to  it  by  the 
blood-stream,  giving  rise  to  animal  heat  and  other  manifestations  of 


12  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

energy ;  and  finally  the  simple  products  of  oxidation  or  chemical 
breakdown  are  carried  to  the  organs  of  excretion  (lung,  skin,  kidney, 
&c.),  where  they  are  discharged  from  the  body. 

A  candle  consists  principally  of  carbon  and  hydrogen  ;  when  it  is 
burnt  the  products  are  carbonic  acid  gas  and  water ;  the  former  may 
be  detected  by  means  of  lime  water,  the  latter,  by  holding  a  dry 
beaker  upside  down  for  a  few  moments  over  the  burning  candle,  when 
the  moisture  will  condense  on  the  cold  glass. 

The  body  is  more  complex  than  a  candle,  but  so  far  as  its  carbon 
and  hydrogen  are  concerned  the  main  products  of  combustion  are 
the  same.  The  carbon  dioxide  is  discharged  by  the  expired  air,  as 
may  be  proved  by  blowing  it  into  lime  water.  The  water  finds  an 
outlet  by  several  channels,  lungs,  skin,  and  kidneys.  The  presence  of 
nitrogen  in  the  body  is  perhaps  the  most  striking  chemical  distinction 
between  it  and  a  candle,  and  here  again  the  process  of  metabolism 
runs  a  course  analogous  in  some  degree  to  our  experiments  in  vitro. 
Here  again  the  most  important  and  abundant  substance  which 
contains  the  waste  nitrogen  is  the  simple  material  ammonia,  but 
ammonia  is  only  discharged  as  such  to  a  very  small  extent  in  health. 
It  unites  with  carbon  and  oxygen  to  form  the  body  called  urea 
(CON2H4),  which  finds  its  way  out  of  the  body  via  the  urine.  The 
urine  also  contains  the  sulphates,  due  to  the  oxidation  of  the  sulphur 
of  the  proteins,  and  the  phosphates  due  to  the  similar  oxidation  of 
the  phosphorus  of  such  substances  as  lecithin  and  nuclein.  Some 
of  the  salts  of  the  urine,  however,  in  particular  the  chlorides,  come 
directly  from  the  food.  This  we  shall  discuss  at  the  proper  place 
when  we  come  to  the  study  of  the  urine. 


LESSON   II 
THE  CARBOHYDRATES 

1.  Note  the  general  appearance  of  the  specimens  of  grape  sugar  or  dextrose, 
cane  sugar,  dextrin,  and  starch  which  are  given  round. 

2.  Put  some  of  each  into  cold  water.  Starch  is  insoluble  ;  dextrose,  cane 
sugar,  and  dextrin  dissolve  after  a  time,  but  more  readily  in  hot  water. 

3.  Trommer's  test. — Put  a  few  drops  of  copper  sulphate  solution  into  a 
test-tube,  then  solution  of  dextrose,  and  then  strong  caustic  potash.  On 
adding  the  caustic  potash  a  precipitate  is  first  formed,  which,  owing  to  the 
presence  of  the  sugar,  rapidly  redissolves,  forming  a  blue  solution.  On  boil- 
ing this  a  yellow  or  red  precipitate  (cuprous  hydrate  or  oxide)  forms. 

4.  Fehling's  test. — Fehling's  solution  is  a  mixture  of  copper  sulphate, 
caustic  soda,  and  Rochelle  salt  of  a  certain  strength.  It  is  used  for  esti- 
mating dextrose  quantitatively  (see  Lesson  XXL).  It  may  be  used  as  a  qualita- 
tive test  also.  Boil  some  Fehling's  solution  ;  if  it  remains  clear  it  is  in  good 
condition ;  add  to  it  an  equal  volume  of  solution  of  dextrose  and  boil  again. 
Reduction,  resulting  in  the  formation  of  cuprous  hydrate  or  oxide,  takes 
place  as  in  Trommer's  test. 

5.  Moore's  test. — Add  to  the  dextrose  solution  about  half  its  volume  of 
20-per-cent.  potash  and  heat.  The  solution  becomes  yellowish  brown.  Add 
to  this  some  sulphuric  acid  (25  per  cent.)  and  the  odour  of  caramel  becomes 
apparent. 

6.  Fermentation  test. — Add  a  fragment  of  dried  yeast  to  the  dextrose 
solution  in  a  test-tube  ;  fill  the  test-tui)e  up  with  mercury,  and  invert  it  over 
mercury  in  a  trough.  Place  it  in  an  incubator  at  body  temperature  for  24 
hours.  The  sugar  is  broken  up  into  alcohol  and  carbon  dioxide ;  the  latter 
gas  collects  in  the  upper  part  of  the  test-tube. 

7.  Ca7ie  Sugar. — (a)  The  solution  of  cane  sugar  when  mixed  with  copper 
sulphate  and  caustic  potash  gives  a  blue  solution.  But  on  boiling  no  reduc- 
tion occurs. 

(6)  Take  some  of  the  cane-sugar  solution  and  boil  it  with  a  few  drops  of 
25-per-cent.  sulphuric  acid.  This  converts  it  into  equal  parts  of 
dextrose  and  levulose.  It  then  gives  Trommer's  or  Fehling's  test 
in  the  typical  way. 

(c)  Boil  some  of  the  cane-sugar  solution  with  an  equal  volume  of  con- 
centrated hydrochloric  acid.  A  deep  red  solutionis  formed.  Dex- 
trose,  lactose,  and  maltose  do  not  give  this  test. 

8.  Starch. — (a)  Examine  microscopically  the  scrapings  from  the  surface 
of  a  freshly  cut  potato.  Note  the  appearance  of  the  starch  grains  with  their 
concentric  markings. 

(6)  On  boiling  starch  with  water  an  opalescent  solution  is  formed,  which, 
if  strong,  gelatinises  on  cooling. 

(c)  Add  iodine  solution.  An  intense  blue  colour  is  produced,  which  dis- 
appears on  heating,  and  if  not  heated  too  long  reappears  on  cooling. 
N.B. — Prolonged  heating  drives  off  the  iodine,  and  consequently 
no  blue  colour  returns  after  cooling. 


14  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

{(J)  Conversion  into  dextrin  and  dextrose.  To  some  starch  solution  in  a 
flask  add  a  few  drops  of  25-per-cent.  sulphuric  acid,  and  boil  for 
15  minutes.  Take  some  of  the  liquid,  which  is  now  clear,  and  show 
the  presence  of  dextrin  and  dextrose. 

9.  Dextrin.  —Add  iodine  solution  to  solution  of  dextrin  ;  a  reddish-brown 
colour  is  produced.  The  colour  disappears  on  heating  and  reappears  on 
coohng. 

10.  Glycogen. — Solution  of  glycogen  is  given  round :  (a)  it  is  opalescent 
like  that  of  starch. 

(6)  With  iodine  solution  it  gives  a  brown  colour  very  like  that  given  by 
dextrin.    The  colour  disappears  on  heating  and  reappears  on  cooling. 

(c)  By  boiling  with  25-per-cent.  sulphuric  acid  for  15  to  20  minutes  it  is 
converted  into  grape  sugar. 

The  carbohydrates  are  found  chiefly  in  vegetable  tissues,  and 
many  of  them  form  important  foods.  Some  carbohydrates  are, 
however,  found  in  or  formed  by  the  animal  organism.  The  most 
important  of  these  are  glycogen,  or  animal  starch ;  dextrose ;  and 
lactose,  or  milk  sugar. 

The  carbohydrates  may  be  conveniently  defined  as  compounds  of 
carbon,  hydrogen,  and  oxygen,  the  two  last  named  elements  being  in 
the  proportion  in  which  they  occur  in  water.  But  this  definition  is 
only  a  rough  one,  and  if  pushed  too  far  would  include  many  substances, 
like  acetic  acid,  lactic  acid,  and  inosite,  which  are  not  carbohydrates. 
Kesearch  has  shown  that  the  chemical  constitution  of  the  simplest 
carbohydrates  is  that  of  an  aldehyde,  or  a  ketone,  and  that  the  more 
complex  carbohydrates  are  condensation  products  of  the  simple  ones. 
In  order,  therefore,  that  we  may  understand  the  constitution  of  these 
substances,  it  is  first  necessary  that  we  should  understand  what  is 
meant  by  the  terms  aldehyde  and  ketone. 

A  primary  alcohol  is  one  in  which  the  hydroxyl  (OH)  is  attached 
to  the  last  carbon  atom  of  the  chain  ;  its  end  group  is  CH2OH.  Thus 
the  formula  for  common  alcohol  (primary  ethyl  alcohol)  is 

CH3.CH2OH. 

The  formula  for  the  next  alcohol  of  the  same  series   (primary 

propyl  alcohol)  is 

0H3.CH,.CH2OH. 

If  a  primary  alcohol  is  oxidised,  the  first  oxidation  product  is 
called  an  aldehyde  ;  thus  ethyl  alcohol  yields  acetic  aldehyde  : — 

CH3.CH2OH  -f  0=CH3.CH0  +  H2O. 

[ethj'l  alcohol]  [acetic  aldehyde] 

The  typical  end-group  CHO  of  the  aldehyde  is  not  stable,  but  is 
easily  oxidisable  to  form  the  group  COOH,  and  the  compound  so 
formed  is  called  an  acid  ;  in  this  way  acetic  aldehyde  forms  acetic 
acid : — 


THE   CARBOHYDEATES  15 

CH3.CHO  +  0=CH3.C00H. 

[acetic  aldehyde]  [acetic  acid] 

The  majority  of  the  simple  sugars  are  aldehydes  of  more  complex 
alcohols  than  this  :  they  are  spoken  of  as  aldoses.  The  readiness  with 
which  aldehydes  are  oxidisable  renders  them  powerful  reducing  agents, 
and  this  furnishes  us  with  some  of  the  tests  for  the  sugars. 

Let  us  now  turn  to  the  case  of  the  ketones.  A  secondary  alcohol 
is  one  in  which  the  OH  group  is  attached  to  a  central  carbon  atom  ; 
thus  secondary  propyl  alcohol  has  the  formula 

CH3.CHOH.GH3. 

Its  typical  group  is  therefore  CHOH.  When  this  is  oxidised,  the 
first  oxidation  product  is  called  a  ketone,  thus  : — 

CH3.CHOH.CH3  +  0=CH3.CO.CH3  + H2O. 

[secondary  propyl  alcohol]  [propyl  ketone] 

It  therefore  contains  the  group  CO  in  the  middle  of  the  chain. 
Some  of  the  sugars  are  ketones  of  more  complex  alcohols  :  these  are 
called  ketoses.  The  only  one  of  these  which  is  of  physiological  interest 
is  levulose. 

The  alcohols  of  which  we  have  already  spoken  are  called  monatomic, 
because  they  contain  only  one  OH  group.  Those  which  contain  two 
OH  groups  (like  glycol)  are  called  diatomic ;  those  which  contain 
three  OH  groups  (like  glycerin)  are  called  triatomic  ;  and  so  on.  The 
hexatomic  alcohols  are  those  which  contain  six  OH  groups.  Three 
of  these  hexatomic  alcohols  with  the  formula  C6H8(OH)(j  are  of 
physiological  interest ;  they  are  isomerides,  and  their  names  are 
sorbite,  mannite,  and  dulcite.  By  careful  oxidation  their  aldehydes 
and  ketones  can  be  obtained  ;  these  are  the  simple  sugars  ;  thus, 
dextrose  is  the  aldehyde  of  sorbite ;  mannose  is  the  aldehyde  of  mannite ; 
levulose  is  the  ketone  of  mannite  ;  and  galactose  is  the  aldehyde  of 
dulcite.  The  sugars  all  have  the  empirical  formula  C^HigOe.  The 
constitutional  formula  for  dextrose  is  : — 

H       H        H        H        H       H 

I          I           I          I          I  I 

H C C C C C C 

I  I  I  II  I 

OH     OH     OH     OH     OH      O 

By  further  oxidation,  the  sugars  yield  acids  with  various  names. 
If  we  take  such  a  sugar  as  a  typical  specimen,  we  see  that  their  general 
formula  is 

C„H2,hO„i 


16 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


and  as  a  general  rule  n—m\  that  is,  the  number  of  oxygen  and  carbon 
atoms  is  equal.  This  number  in  the  case  of  the  sugars  already 
mentioned  is  six.     Hence  they  are  called  hexoses. 

Sugars  are  known  to  chemists,  in  which  this  number  is  3, 4,  5,  7,  &c.,  and 
these  are  called  trioses,  tetroses,  pentoses,  heptoses,  &c.  The  majority  of 
these  have  no  physiological  interest.  It  should,  however,  be  mentioned  that 
a  pentose  has  been  obtained  from  the  nucleoprotein  of  the  pancreas,  of  the 
liver,  and  of  yeast.  If  the  pentoses  that  are  found  in  various  plants  are  given 
to  an  animal,  they  are  excreted  in  great  measure  unchanged  in  the  urine. 

The  hexoses  are  of  great  physiological  importance.  The  prin- 
cipal ones  are  dextrose,  levulose,  and  galactose.  These  are  called 
monosaccharides. 

Another  important  group  of  sugars  is  that  of  the  disaccharides : 
these  are  formed  by  the  combination  of  two  molecules  of  monosac- 
charide together  with  the  loss  of  a  molecule  of  water,  thus  : — 

C5Hi20o  +  C6Hi206  =  Ci2H220ii-f-H20. 

The  principal  members  of  the  disaccharide  group  are  cane  sugar, 
lactose,  and  maltose. 

If  more  than  two  molecules  of  the  monosaccharide  group  undergo 
a  corresponding  condensation,  we  get  what  are  called  polysaccharides. 

The  polysaccharides  are  starch,  glycogen,  various  dextrins,  cellu- 
lose, gums,  &c.  We  may  therefore  arrange  the  important  carbo- 
hydrates of  the  hexose  family  in  a  tabular  form  as  follows  : — 


1.  Monosaccharides  or  Glucoses, 

2.  Disaccharides,  Sueroses, 
or  Saccharoses,  CiaHjaO,, 

3.  Polysaccharides  or  Amyloses 

+  Dextrose. 
—  Levulose. 
+  Galactose. 

+  Cane  sugar. 
+  Lactose. 
+  Maltose. 

+  Starch. 
+  Glycogen. 
+  Dextrin. 
Cellulose. 

The  signs  +  and  —  in  the  above  list  indicate  that  the  substances 

to  which  they  are  prefixed  are  dextro-  and  levo-rotatory  respectively 

as  regards  polarised  light.  ^      The  formulae  given  above  are  merely 

empirical;  the  quantity  w  in  the  starch  gi'oup  is  variable  and  often 

large.     The  following  are  the  chief  facts  in  relation  to  each  of  the 

principal  carbohydrates. 

'  For  a  description  of  polarised  light  and  polarimeters  see  Appendix.  This 
and  the  other  matter  in  the  Appendix  are  placed  there  for  convenience,  not  because 
they  are  unimportant.  Students  are  therefore  urged  to  refer  to  and  carefully  study 
these  subjects. 


THE   CAKBOHYDRATES  17 

MONOSACCHARIDES 

Dextrose  or  Grape  Sugar. — This  carbohydrate  is  found  in  fruits, 
honey,  and  in  minute  quantities  in  the  blood  (0'12  per  cent.)  and 
numerous  tissues,  organs,  and  fluids  of  the 
body.  It  is  the  form  of  sugar  found  in 
large  quantities  in  the  blood  and  urine 
in  the  disease  known  as  diabetes. 

Dextrose  is  soluble  in  hot  and  cold 
water  and  in  alcohol.  It  is  crystalline 
(see  fig.  1),  but  not  so  sweet  as'cane  sugar. 
When  heated  with  strong  potash  certain 
complex  acids  are  formed  which  have  a 
yellow  or  brown  colour.    This  constitutes 

Moore's  test  ior  SUgSiT.       In  alkaline  SOlu-  Fig.  l. -Dextrose  crystals. 

tions   dextrose   reduces   salts   of    silver, 

bismuth,  mercury,  and  copper.  The  reduction  of  cupric  hydrate  to 
cuprous  hydrate  or  oxide  constitutes  Trommer's  test,  which  has  been 
already  described  at  the  head  of  the  lesson.  On  boiling  it  with  an 
alkaline  solution  of  picric  acid,  a  dark  red  opaque  solution  due  to  reduc- 
tion of  the  picric  to  picramic  acid  is  produced.  Another  important 
property  of  grape  sugar  is  that  under  the  influence  of  yeast  it  is  con- 
verted into  alcohol  and  carbonic  acid  (C6Hi206=2C2H60  +  2C02). 

Dextrose  may  be  estimated  by  the  fermentation  test,  by  the 
polarimeter,  and  by  the  use  of  Fehling's  solution.  The  last  method 
is  the  most  important :  it  rests  on  the  same  principles  as  Trommer's 
test,  and  we  shall  study  it  and  other  methods  of  estimating  sugar  in 
connection  with  diabetic  urine  (see  Lesson  XII.). 

Levulose. — When  cane  sugar  is  treated  with  dilute  mineral  acids 
it  undergoes  a  process  known  as  inversion — i.e.  it  takes  up  water  and 
is  converted  into  equal  parts  of  dextrose  and  levulose.  The  pre- 
viously dextro-rotatory  solution  of  cane  sugar  then  becomes  levo- 
rotator}^  the  levo -rotatory  power  of  the  levulose  being  greater  than 
the  dextro-rotatory  power  of  the  dextrose  formed.  Hence  the  term 
inversio7i.  The  same  hydrolytic  change  is  produced  by  certain 
ferments,  such  as  the  invert  ferment  of  the  intestinal  juice,  and  of 
yeast. 

Pure  levulose  can  be  crystallised,  but  so  great  is  the  difiSculty  of 
obtaining  crystals  of  it  that  one  of  its  names  was  '  uncrystallisable 
sugar.'  Small  quantities  of  levulose  have  been  found  in  blood,  urine, 
and  muscle.  It  has  been  recommended  as  an  article  of  diet  in 
diabetes   in   place   of   ordinary   sugar;    in  this  disease  it  does  not 

c 


18  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

appear  to  have  the  harmful  effect  that  other  sugars  produce.     Levu- 

lose  gives  the  same  general  reactions  as  dextrose. 

Galactose   is   formed  by  the   action   of  dilute   mineral   acids   or 

inverting  ferments  on  lactose  or 
milk  sugar.  It  resembles  dextrose 
in  being  dextro-rotatory,  in  reducing 
cupric  hydrate  in  Trommer's  test, 
and  in  being  directly  fermentable 
with  yeast.  When  oxidised  by  means 
of  nitric  acid  it,  however,  yields  an 
acid  called  mucic  acid  (CgHioOg), 
which  is  only  sparingly  soluble  in 
water.  Dextrose  when  treated  in  this 
way  yields  an  isomeric  acid — i.e.  an 
acid  with  the  same  empirical  formula, 
called  saccharic  acid,  which  is  readily 
2.-inosite  crystals.  soluble  in  Water. 

Inosite,  formerly  called  muscle  sugar,  is  found  in  muscle,  kidney,  liver, 
and  other  parts  of  the  body  in  small  quantities.  It  is  also  largely  found  in 
the  vegetable  kingdom.  It  is  a  crystallisable  substance  (see  fig.  2)  and  has 
the  same  formula  (C,.Hj.^O,;)  as  the  glucoses.  It  is,  however,  not  a  sugar. 
It  gives  none  of  the  sugar  tests,  and  careful  analysis  has  shown  it  has  quite 
a  different  chemical  constitution  from  the  true  sugars.  It  belongs  to  the 
aromatic  series,  and  is  only  included  here  for  convenience. 

DISACCHAEIDES 

Cane  Sugar. — This  sugar  is  generally  distributed  throughout  the 
vegetable  kingdom  in  the  juices  of  plants  and  fruits,  especially  the 
sugar  cane,  beetroot,  mallow,  and  sugar  maple.  It  is  a  substance  of 
great  importance  as  a  food.  After  abundant  ingestion  of  cane  sugar 
traces  may  appear  in  the  urine,  but  the  greater  part  undergoes 
inversion  in  the  alimentary  canal. 

Pure  cane  sugar  is  crystalline  and  dextro-rotatory.  It  holds  cupric 
hydrate  in  solution  in  an  alkaline  liquid — tlcfkt  is,  with  Trommer's 
test  it  gives  a  blue  solution.  But  no  reduction  occurs  on  boiling. 
After  inversion  it  is  strongly  reducing. 

Inversion  may  be  brought  about  readily  by  boiling  with  dilute 
mineral  acids,  or  by  means  of  an  inverting  ferment,  such  as  that 
occurring  in  the  succus  entericus  or  intestinal  juice.  It  then  takes 
up  water  and  is  split  into  equal  parts  of  dextrose  and  levulose  : — 

Cj2H220ii+H20  =  C6H,206  +  C(,H,206 

[caoe  sugar]  [dextrose]  [levulose] 


THE  CARBOHYDRATES  19 

With  yeast,  cane  sugar  is  first  inverted  by  means  of  a  special 
soluble  ferment  produced  by  the  yeast  cells,  and  then  there  is  an 
alcoholic  fermentation  of  the  glucoses  so  formed. 

Lactose,  or  milk  sugar,  occurs  in  milk.  It  has  also  been  described 
as  occurring  in  the  urine  of  women  in  the  early  days  of  lactation  or 
after  weaning. 

It  crystallises  in   rhombic  prisms  (see  fig.  3).     It  is   much   less 
soluble  in  water  than  cane  sugar  or  dextrose, 
and  has  only  a  slightly  sweet  taste.     It  is 
insoluble   in   alcohol  and   ether;    aqueous 
solutions  are  dextro-rotatory. 

Solutions  of  lactose  give  Trommer's 
test,  but  when  the  reducing  power  is  tested 
quantitatively  by  Fehling's  solution  it  is 
found  to  be  a  less  powerful  reducing  agent  ^  / 

than  dextrose.     If   it  required   seven  parts        fig.  3.— Miik-sugar  crystals. 
of  a  solution  of  dextrose  to  reduce  a  given 

quantity  of  Fehling's  solution,  it  would  require  ten  parts  of  a  solution 
of  lactose  of  the  same  strength  to  reduce  the  same  quantity  of 
Fehling's  solution. 

Lactose,  like  cane  sugar,  can  be  hydrolysed  by  the  same  agencies 
as  those  already  enumerated  in  connection  with  cane  sugar.  The 
glucoses  formed  are  dextrose  and  galactose, 

C,2H2,OH  +  H2O=C6Hi2Oe  +  0eH,A 

[lactose]  [dextrose]  [galactose] 

With  yeast  it  is  first  inverted,  and  then  alcohol  is  formed.  Thi^, 
however,  occurs  slowly. 

With  the  lactic-acid  organisms  which  bring  about  the  souring  of 
milk  the  lactic-acid  fermentation  is  produced.  This  may  also  occur 
as  the  result  of  the  action  of  putrefactive  bacteria  in  the  alimentary 
canal.  The  two  stages  of  the  lactic -acid  fermentation  are  represented 
by  the  following  equations  : — 

(1)  C,,B.,,0,,-\-IL,0=:iG,R,0, 

[lactose]  [lactic  acid] 

(2)  4C3He03=2C4H802-H4C02  +  4H2 

[lactic  acid]      [butyric  acid] 

Maltose  is  the  chief  end  product  of  the  action  of  malt  diastase  on 
starch,  and  is  also  formed  as  an  intermediate  product  in  the  action 
of  dilute  sulphuric  acid  on  the  same  substance.  It  is  also  the  chief 
sugar  formed  from  starch  by  the  diastatic  ferments  contained  in  the 
saliva  (ptyalin)  and  pancreatic  juice  (atnylopsin).     It  can  be  obtained 

c3 


20 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


in  form  of  acicular  crystals  ;  it  is  strongly  dextro-rotatory.  It  gives 
Trommer's  test ;  but  its  reducing  power,  as  measured  by  Fehling's 
solution,  is  one-third  less  than  that  of  dextrose. 

By  prolonged  boiling  with  water,  or,  more  readily,  by  boiling  with 
a  dilute  mineral  acid,  or  by  means  of  an  inverting  ferment,  such  as 
occurs  in  the  intestinal  juice,  it  is  converted  into  dextrose. 

0i2H22Oh+H2O  =  C6H.2O6  +  C6Hp,O6 

[maltose]  [dextrose]        [dextrose] 

It  undergoes  readily  the  alcoholic  fermentation. 

The  three  important  physiological  sugars  (dextrose,  lactose,  and 
maltose)  may  be  distinguished  from  one  another  by  their  relative 
reducing  action  on  Fehling's  solution  (I'0 :  071 :  0-63),  by  their 
rotatory  power,  or  by  the  phenyl-hydrazine  test  described  in  Lesson 
XIII. 

POLYSACCHARIDES 

Starch,  is  widely  diffused  through  the  vegetable  kingdom.  It 
occurs  in  nature  in  the  form  of  microscopic  grains,  varying  in  size 
and  appearance,  according  to  their  source.  Each  consists  of  a 
central  spot  (hilum)  round  which  more  or  less  concentric  envelopes 

of  starch  proper  or  granulose  alternate 
with  layers  of  cellulose.  Cellulose  has 
very  little  digestive  value,  but  starch  is 
a  most  important  food. 

Starch  is  insoluble  in  cold  water  :    it 
forms  an  opalescent  solution  in  boiling 
water,  which  if  concentrated  gelatinises 
on  cooling.     Its  most  characteristic  re- 
^\mmmsr^smB^^mmm^<^        action  is  the  blue   colour  it  gives  with 
i^^BSPf^P^H^         iodine  solution. 

•'^^^^^^^^^^^  On  heating  starch  with  dilute  mineral 

acids  dextrose  is  formed.  By  the  action 
of  diastatic  ferments,  maltose  is  the  chief 
end  product.  In  both  cases  dextrin  is 
an  intermediate  stage  in  the  process. 
Before  the  formation  of  dextrin  the  starch  solution  loses  its  opal- 
escence, a  substance  called  soluble  starch  or  amidulin  being  formed. 
This,  like  native  starch,  gives  a  blue  colour  with  iodine  solution. 
Although  the  molecular  weight  of  starch  is  unknown,  the  formula 
for  soluble  starch  is  probably  (CgH, 005)200-  Equations  that  repre- 
sent the  formation  of  sugars  and  dextrin s  from  this  are  very  complex, 
and  are  at  present  hypothetical. 


Fig.  4.-  Section  of  pea  showing  starch 
aud  aleurone  grains  embedded  in  the 
protoplasm  of  the  cells  :  a,  aleurone 
grains ;  s(,  starch  grains ;  i,  inter- 
cellular spaces.    (Yeo,  after  Sachs.) 


THE   CAKBOHYDRATES  21 

Dextrin  is  the  name  given  to  the  intermediate  products  in  the 
hydrolysis  of  starch,  and  two  chief  varieties  are  distinguished — 
erythro-dextrin,  v^hich  gives  a  reddish-brown  colour  with  iodine  solu- 
tion ;  and  achroo-dextrin,  which  does  not.  v 

It  is  readily  soluble  in  water,  but  insoluble  in  alcohol  and  ether. 
It  is  gummy  and  amorphous.  It  does  not  give  Trommer's  test,  nor 
does  it  ferment  with  yeast.  It  is  dextro-rotatory.  By  hydrating 
agencies  it  is  converted  into  glucose. 

Glycogen,  or  animal  starch,  is  found  in  liver,  muscle,  colourless 
blood  corpuscles  and  other  tissues. 

Glycogen  is  a  white  tasteless  powder,  soluble  in  water,  but  it 
forms,  like  starch,  an  opalescent  solution.  It  is  insoluble  in  alcohol 
and  ether.  It  is  dextro-rotatory.  With  Trommer's  test  it  gives  a 
blue  solution,  but  no  reduction  occurs  on  boiling. 

With  iodine  solution  it  gives  a  reddish  or  port-wine  colour,  very 
similar  to  that  given  by  erythro-dextrin.  Dextrin  may  be  distin- 
guished from  glycogen  by  (1)  the  fact  that  it  gives  a  clear,  not  an 
opalescent,  solution  with  water ;  and  (2)  it  is  not  precipitated  by  basic 
lead  acetate  as  glycogen  is.  It  is,  however,  precipitated  by  basic  lead 
acetate  and  ammonia.  (3)  Glycogen  is  precipitated  by  55  per  cent. 
of  alcohol ;  the  dextrins  require  85  per  cent,  or  more. 

Cellulose. — This  is  the  colourless  material  of  which  the  cell-walls 
and  woody  fibres  of  plants  are  composed.  By  treatment  with 
strong  mineral  acids,  it  is  like  starch,  converted  into  glucose,  but 
with  much  greater  difficulty.  The  various  digestive  ferments  have 
little  or  no  action  on  cellulose  ;  hence  the  necessity  of  boiling  starch 
before  it  is  taken  as  food.  Boiling  bursts  the  cellulose  envelope  of 
the  starch  grains,  and  so  allows  the  digestive  juice  to  get  at  the 
starch  proper.  Cellulose  is  found  in  a  few  animals,  as  in  the  test  or 
outer  investment  of  the  Tunicates. 

Salting  out  of  the  Colloid  Carbohydrates. — By  saturating  solutions  of 
the  colloid  carbohydrates  (starch,  soluble  starch,  glycogen,  and  some  varieties 
of  dextrin)  with  such  neutral  salts  as  magnesium  sulphate  or  ammonium 
sulphate  the  carbohydrate  is  thrown  out  of  solution  in  the  form  of  a  white 
precipitate.  The  remaining  carbohydrates  (sugars  and  some  of  the  smaller 
moleculed  dextrins  like  achroo-dextrin)  are  not  precipitated  by  this  means. 
We  shall  find  in  connection  with  the  proteins  that  this  method,  known  as 
*  salting  out,'  is  one  largely  employed  there  for  precipitating  and  distinguish- 
ing between  classes  of  proteins.  The  student  is  therefore  warned  that  a 
precipitate  obtained  under  such  circumstances  will  not  necessarily  indicate 
the  presence  of  protein. 

Further  information  regarding  the  carbohydrates  is  given  in  I^esson  XIII, 


LESSON   III 

THE   FATS 

Lard  and  olive  oil  are  given  round  as  examples  of  fats. 

1.  They  are  insoluble  in  water. 

2.  They  dissolve  readily  in  ether.  On  pouring  some  of  the  ethereal  solu- 
tion on  to  a  piece  of  blotting  paper,  a  greasy  stain  is  left  after  the  ether  has 
evaporated. 

3.  Saponification. — By  boiling  with  potash,  fat  yields  a  solution  of  soap. 
On  adding  some  sulphuric  acid  to  this  and  heating,  the  fatty  acid  collects  in 
a  layer  on  the  surface  of  the  fluid.  This  experiment  may  conveniently  be 
performed  in  the  following  way :  —Melt  some  lard  in  an  evaporating  basin 
and  pour  it  into  a  solution  of  potash  in  alcohol  -  contained  in  a  small  flask 
and  heated  carefully  on  a  water-hath  nearly  to  boiling-point.  Continue  to 
boil  and  saponification  is  soon  completed.  When  the  process  is  completed 
drop  some  of  the  solution  into  a  test-tube  containing  about  10  c.c.  of  water ; 
the  solution  of  soap  will  be  clear,  and  no  oil  globules  should  separate  out. 
If  there  is  any  separation  of  oil  globules  continue  the  boiling. 

Then  drop  the  soap  solution  into  some  25-per-cent.  sulphuric  acid  con- 
tained in  a  small  beaker  and  heated  nearly  to  boiling ;  the  fatty  acid  soon 
separates  out  and  floats  on  the  surface. 

4.  Beaction  of  Fatty  Acids. — (a)  They  produce  a  greasy  stain  on  paper. 
(b)  Wash  the  fatty  acid  obtained  in  experiment  3  repeatedly  with  water, 

rmtil  the  wash  water  is  no  longer  acid,  and  divide  it  into  two  portions. 
Dissolve  one  portion  in  ether ;  this  solution  reacts  acid  to  phenolphthalein  ; 
to  show  this,  place  a  few  drops  of  phenolphthalein  in  5  c.c.  of  water  contain- 
ing a  drop  of  20-per-cent.  potash.  If  this  red  solution  is  dropped  into  the 
solution  of  fatty  acid,  the  colour  is  discharged.  Place  the  second  portion  of 
fatty  acid  in  some  half-saturated  solution  of  sodium  carbonate  and  warm ;  a 
solution  of  sodium  soap  is  obtained  and  carbon  dioxide  comes  ofl". 

5.  Separation  of  Neutral  Fats  from  Fatty  Acids. — In  most  fats  some 
free  fatty  acid  is  present.  They  may  be  separated  by  the  fact  that  the  latter 
only  dissolves  in  a  solution  of  sodium  carbonate  to  form  soap.  The  resulting 
mass  is  shaken  with  water  and  then  with  ether ;  the  two  fluids  separate  on 
standing  ;  the  ether  contains  the  neutral  fat  and  the  water  the  soap. 

6.  Test  for  Glycerin. — The  most  important  reaction  for  glycerin,  the 
other  constituent  of  a  fat,  is  the  acrolein  test,  which  is  performed  in  the 
following  way : — Place  some  lard  in  a  dry  test-tube,  add  a  crystal  of  potassium 
sulphate  and  heat.  Acrolein  is  given  off,  which  is  recognised  by  its  charac- 
teristic unpleasant  odour,  and  by  the  fact  that  it  blackens  a  piece  of  filter 
paper  previously  moistened  with  ammoniacal  silver  nitrate  solution. 

7.  Osmic  Acid  Test. — Fat,  if  it  contains  olein  or  oleic  acid,  is  blackened 
by  osmic  acid.     Try  this  with  both  the  lard  and  the  olive  oil. 

'  30  grammes  of  potash  are  dissolved  in  20  c.c.  of  water,  and  200  c.c.  of  90-per- 
cent, alcohol  added. 


THE  FATS 


23 


8.  To  determine  melting-point  of  a  fat  or  fatty  acid,  place  a  small 
quantity  in  a  very  narrow  test-tube  strapped  on  to  a  thermometer  with  an 
india-rubber  band.  Place  this  in  a  water-bath  which  is  gradually  heated, 
and  note  the  temperature  at  which  it  melts. 

9.  Emulsijication. — (a)  Take  two  test-tubes  and  label  them  A  and  B. 
Place  water  in  A  and  soap  solution  in  B.  To  each  add  a  few  drops  of  olive 
oil  and  shake.     In  B  an  emulsion  is  formed,  but  not  in  A. 

(6)  Shake  a  few  drops  of  rancid  oil  with  a  dilute  solution  of  potash ;  an 
emulsion  is  formed  because  the  potash  and  free  fatty  acid  unite  to  form  a  soap. 
Divide  this  into  two  parts,  and  to  one  of  them  add  a  little  solution  of  gum  or 
egg  albumin ;  the  emulsion  is  much  more  permanent  in  this  specimen. 
These  experiments  illustrate  the  favourable  action  of  soap  and  of  a  suspend- 
ing medium  like  mucilage  upon  the  formation  of  an  emulsion. 

Fat  is  found  in  small  quantities  in  many  animal  tissues.  It  is, 
hov^ever,    found    in    large  quantities  in  three  situations,   viz.   bone 


Fia.  5.— A  few  cells  from  the  margin  of  a  fat  lobule  ;  /.  g.,  fat  globule  distending  fat  cell ;  ??,  nucleus  ; 
m,  membranous  envelope  of  tlie  cell  ;  cr.,  bunch  of  crystals  within  a  fat  cell ;  c,  capillary  vessel 
V,  venule ;  c.  t.,  connective  tissue  cell.    The  fibres  of  the  connective  tissue  are  not  represented. 


marrow,  adipose  tissue,  and  milk.     The  consideration  of  the  fat  in 
milk  is  postponed  to  Lesson  VI. 

The  contents  of  the  fat  cells  of  adipose  tissue  are  fluid  during  life, 
the  normal  temperature  of  the  body  (37°  C,  or  99°  F.)  being  con- 
siderably above  the  melting-point  (25°  C.)  of  the  mixture  of  the  fats 
found  there.  These  fats  are  three  in  number,  and  are  called  palmitin, 
stearin,  and  olein.  They  differ  from  one  another  in  chemical  com- 
position and  in  certain  physical  characters,  such  as  melting-point 
and  solubilities.  Olein  melts  at  —5°  C,  palmitin  at  45°  C,  and  stearin 
at  53-65°  C.     Thus,  it  is  olein  v^hich  holds  the  other  tv^o  dissolved  at 


24  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

the  body  temperature.  Fats  are  all  soluble  in  hot  alcohol,  ether,  and 
chloroform,  but  insoluble  in  water. 

Chemical  Constitution  of  the  Fats. — The  fats  are  compounds  of 
fatty  acids  with  glycerin,  and  may  be  termed  glycerides  or  glyceric 
ethers. 

The  fatty  acids  form  a  series  of  acids  derived  from  the  mona- 
tomic  alcohols  by  oxidation.  Thus,  to  take  ordinary  ethyl  alcohol, 
C2H5HO,  the  first  stage  in  oxidation  is  the  removal  of  two  atoms 
of  hydrogen  to  form  aldehyde,  CH3.COH  :  on  further  oxidation  an 
atom  of  oxygen  is  added  to  form  acetic  acid,  CH3.COOH  (see  also 
p.U). 

A  similar  acid  can  be  obtained  from  all  the  other  alcohols, 
thus: 

From  methyl  alcohol  CH3.HO,   formic   acid   H.COOH   is  obtained 
ethyl 


CH3.HO,   formic   acid  H.COOH   is 

C2H5.HO,  acetic       „  CH3.COOH 

C3H7.HO,  propionic,,  C^H^.COOH 

butyl  „        C4H.J.HO,  butyric     „  C3H7.COOH 

amyl  „        CsHn-HO,  valeric   „  C4Hc).C00H 

hexyl         „         C6H,3.HO,caproic  „  GsHn-COOH 


propyl 
butyl 


and  so  on. 

Or  in  general  terms  : — 

From  the  alcohol  with  formula  C,iH2„+i.H0  the  acid  with  formula 
C„_iH2„_i.C0.0H  is  obtained.  The  sixteenth  term  of  this  series  has 
the  formula  Ci5H3,.C0.0H,  and  is  called  palmitic  acid;  the 
eighteenth  has  the  formula  Ci;H35.CO.OH,  and  is  called  stearic  acid. 
Each  acid,  as  will  be  seen,  consists  of  a  radical,  C„_,H2„_iC0,  united 
to  hydroxyl  (HO). 

Oleic  acid,  however,  is  not  a  member  of  the  fatty  acid  series 
proper,  but  belongs  to  a  somewhat  similar  series  of  acids  known  as 
the  acrylic  series,  of  which  the  general  formula  is  C„_]H2„__3COOH. 
It  is  the  eighteenth  term  of  the  series,  and  its  formula  is  C17H33.CO.OH. 

The  first  member  of  the  group  of  alcohols  from  which  this  acrylic  series  of 
acids  is  obtained  is  called  allyl  alcohol  (CH2 :  CH.CHoOH) ;  the  aldehyde  of 
this  is  acrolein  (CH2  :  CH.CHO),  and  the  formula  for  the  acid  (acrylic  acid)  is 
CH2 :  CH.COOH.  It  will  be  noticed  that  two  of  the  carbon  atoms  are  united 
by  two  valencies,  and  these  bodies  are  therefore  unsaturated ;  they  are  un- 
stable and  are  prone  to  undergo  by  uniting  with  another  element  a  conversion 
into  bodies  in  which  the  carbon  atoms  are  united  by  one  bond  only.  This 
accounts  for  their  reducing  action,  and  it  is  owing  to  this  construction  that 
the  colour  reactions  with  osmic  acid  and  Sudan  III.  are  due.  Fat  which 
contains  any  member  of  the  acrylic  series  like  oleic  acid  blackens  osmic  acid, 
by  reducing  it  to  a  lower  (black)  oxide.  Fats  like  palmitin  and  stearin  do 
not  give  this  reaction. 


THE   FATS  25 

Glycerin  or  Glycerol  is  a  triatomic  alcohol,  C3H.5(OH)3 — i.e.  three 
atoms  of  hydroxyl  united  to  a  radical  glyceryl  (C3H5).  The  hydrogen 
in  the  hydroxyl  atoms  is  replaceable  by  other  organic  radicals.  As 
an  example  take  the  radical  of  acetic  acid  called  acetyl  (CH3.CO). 
The  following  formulae  represent  the  derivatives  that  can  be  obtained 
by  replacing  one,  two,  or  all  three  hydroxyl  hydrogen  atoms  in  this 
way  :— 

(OH  (OH  (OH  (O.CH3.CO 

C3H5    OH  C3H5  -I  OH  C3H5    O.CH3.CO  C3H5    O.CH3.CO 

tOH  iO.CH3.CO  lO.CH3.CO  lo.CH3.CO 

[glycerin]  [monoacetin]  [cliacetin]  [triacetin] 

Triacetin  is  a  type  of  a  neutral  fat ;  stearin,  palmitin,  and  olein 
ought  more  properly  to  be  called  tristearin,  tripalmitin,  and  triolein 
respectively.  Each  consists  of  glycerin  in  which  the  three  atoms  of 
hydrogen  in  the  hydroxyls  are  replaced  by  radicals  of  the  fatty  acid. 
This  is  represented  in  the  following  formulae  : — 

Acid  Radical  Fat 

Palmitic  acid  Ci^Hgi.COOH  Palmityl  Ci,H3,.C0  Palmitin  CgH^COCi^Hgi.CO)., 
Stearic  acid  Ci^Hg^.COOH  Stearyl  Ci.Hgg.CO  Stearin  C3H,(OCi,H3,.CO)3 
Oleic  acid        Ci.Hgg.COOH  Oleyl        Cj-Hgg.CO  Olein        C^B..^{OC,^'R^^.CO)^ 

Decomposition  Products  of  the  Fats. — The  fats  split  up  into  the 
substances  out  of  which  they  are  built  up. 

Under  the  influence  of  superheated  steam,  mineral  acids,  and  in 
the  body  by  means  of  certain  ferments  (for  instance,  the  fat-splitting 
ferment  steapsin  of  the  pancreatic  juice),  a  fat  combines  with  water 
and  splits  into  glycerin  and  the  fatty  acid.  The  following  equation 
represents  what  occurs  in  a  fat,  taking  tripalmitin  as  an  example :  — 

C3H5(O.C,5H3iCO)3  +  3H20=C3H5(OH)3  +  3C,5H3iCO.OH 

[palmitin— a  fat]  [glycerin]  [palmitic  acid— a  fatty  acid] 

In  the  process  of  saponification,  much  the  same  sort  of  reaction 
occurs,  the  final  products  being  glycerin  and  a  compound  of  the  base 
with  the  fatty  acid,  which  is  called  a  soap.  Suppose,  for  instance, 
that  potassium  hydrate  is  used  ;  we  get — 

C3H5(O.Ci5H3iCO)3  +  3KHO=C3H5(OH)3  +  3C,5H3iCO.OK 

[palmitin— a  fat]  [glycerin]  [potassium  palmitate— a  soap] 

Emulsification. — Another  change  that  fats  undergo  in  the  body  is 
very  different  from  saponification.  Ifc  is  a  physical  rather  than  a 
chemical  change ;  the  fat  is  broken  up  into  very  small  globules,  such 
as  are  seen  in  the  natural  emulsion — milk. 

Lecithin    (C42H84NPO9).— This   is    a   very   complex   fat,    which 


26  ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 

yields  on  decomposition  not  only  glycerin  and  a  fatty  acid, 
but  phosphoric  acid,  and  an  alkaloid  [N.(CH3)3C2H602]  called 
choline  in  addition.  Lecithin  is  found  to  a  great  extent  in  the 
nervous  system,^  and  to  a  small  extent  in  bile.  Together  with 
cholesterin,  a  crystallisable,  monatomic  alcohol  (C27H45.HO)  which 
we  shall  consider  more  at  length  in  connection  with  the  bile,  it  is 
found  in  small  quantities  in  the  protoplasm  of  all  cells. 

'  See  further  under  Nervous  Tissues,  Lesson  XXII. 


LESSON   IV 
THE  PROTEINS 

1.  Tests  for  Proteins. — The  following  tests  are  to  be  tried  with  a  mixture  of 
one  part  of  white  of  egg  to  ten  of  water.  (Egg-white  contains  a  mixture  of 
albumin  and  globulin.) 

(a)  Heat  Coagulation. — Faintly  acidulate  with  a  few  drops  of  2-per-cent. 
acetic  acid  and  boil.     The  protein  is  rendered  insoluble  (coagulated  protein). 

(6)  Precipitation  ivitJi  Nitric  Acid. — The  addition  of  strong  nitric  acid  to 
the  original  solution  also  produces  a  white  precipitate. 

(c)  XantJioproteic  Reaction. — On  boiling  the  white  precipitate  produced 
by  nitric  acid  it  turns  yellow  ;  after  cooling  add  ammonia  ;  the  yellow  becomes 
orange. 

[d)  Millon's  Test. — Millon's  reagent  (which  is  a  mixture  of  the  nitrates 
of  mercury  containing  excess  of  nitric  acid  ;  see  p.  5)  gives  a  white  precipitate, 
which  turns  brick-red  on  boiling. 

{e)  After  the  addition  of  a  few  drops  of  20-per-cent.  acetic  acid,  potassium 
ferrocyanide  gives  a  white  precipitate. 

(/)  Rose's  or  PiotroiusM's  Test. — Add  one  drop  of  a  1-per-cent.  solution 
of  cupric  sulphate  to  the  original  solution  and  then  caustic  potash,  and  a 
violet  solution  is  obtained. 

Eepeat  experiment  (/)  with  a  solution  of  commercial  peptone,  and  note 
that  a  rose-red  solution  is  obtained.     This  is  called  the  biuret  reaction. 

(g)  Rosenheim's  Formaldehyde  Reactio?!. — Add  to  the  solution  of  com- 
mercial peptone  a  very  dilute  solution  of  formaldehyde  (1 :  2,500),  and  then 
aboiit  one  third  of  the  volume  of  strong  sulphuric  acid  containing  (as  most 
commercial  specimens  of  the  acid  do)  a  trace  of  an  oxidising  agent  such  as 
ferric  chloride  or  nitrous  acid.  A  purple  ring  develops  at  the  surface  of  contact. 
This  reaction  probably  plays  a  part  in  the  original 

(h)  Adamliiewicz  Reaction^  in  which  glacial  acetic  acid  was  used  instead 
of  the  formaldehyde.  Most  commercial  specimens  of  glacial  acetic  acid  con- 
tain hydrogen  peroxide  as  an  impurity  ;  the  oxidising  action  of  this  on  the 
acetic  acid  leads  to  the  formation  of  traces  of  glyoxylic  acid  and  formalde- 
hyde ;  the  necessary  factors  for  the  occurrence  of  the  formaldehyde  reaction 
are  thus  present.  (According  to  Hopkins  glyoxylic  acid  itself  with  pure 
sulphuric  acid  gives  the  test  with  proteins.) 

The  same  reactions  {g  and  h)  are  given  by  the  solution  of  egg-white,  but 
not  so  markedly. 

2.  Action  of  Neutral  Salts. — {a)  Saturate  the  solution  of  egg-white  with 
magnesium  sulphate  by  adding  crystals  of  the  salt  and  grinding  it  up 
thoroughly  in  a  mortar.  A  white  precipitate  of  egg-globulin  is  produced. 
Filter.  The  filtrate  contains  egg-albumin.  The  precipitate  of  the  globulin 
is  very  small. 

(6)  Half  saturate  the  solution  of  egg-white  with  ammonium  sulphate. 
This  may  be  done  by  adding  to  the  solution  an  equal  volume  of  a  saturated 


28  ESSENTIALS   OF   CHEMICAL  PHYSIOLOaY 

solution  of  ammonium  sulphate.     The  precipitate  produced  consists  of  the 
globulin  ;  the  albumin  remains  in  solution. 

(c)  Completely  saturate  another  portion  with  ammonium  sulphate  by 
adding  crystals  of  the  salt  and  grinding  in  a  mortar  — a  precipitate  is  pro- 
duced of  both  the  globulin  and  albumin.  Filter.  The  filtrate  contains  no 
protein. 

(d)  Repeat  the  last  experiment  (c)  with  a  solution  of  commercial  peptone. 
A  precipitate  is  produced  of  the  proteoses  it  contains.  Filter.  The  filtrate 
contains  the  true  peptone.  This  gives  the  biuret  reaction  (see  above),  but 
large  excess  of  strong  potash  must  be  added  on  account  of  the  presence  of 
ammonium  sulphate.  Ammonium  sulphate  added  to  saturation  preci;pitates 
all  proteins  except  peptone. 


LESSON  V 

THE  PROTEINS   {continued) 

1. — Action  of  Acids  and  Alkalis  07i  Albumin. — Take  three  test-tubes  and 
label  them  A,  B,  and  C. 

In  each  place  an  equal  amount  of  diluted  egg-white,  similar  to  that  used 
in  the  last  lesson. 

To  A  add  a  few  drops  of  0*l-per-cent.  solution  of  caustic  potash. 

To  B  add  the  same  amount  of  0'1-per-cent.  solution  of  caustic  potash. 

To  C  add  a  rather  large  amount  of  0*l-per-cent.  sulphuric  acid. 

Put  all  three  into  the  warm  bath  ^  at  about  the  temperature  of  the  body 
(36-40°  C). 

After  five  minutes  remove  test-tube  A,  and  boil.  The  protein  is  no 
longer  coagulated  by  heat,  having  been  converted  into  alkali- albumin.    After 


Fig.  6.— Simple  warm  batli,  as  described  iu  footnote. 

cooling,  colour  with  litmus  solution  and  neutralise  with  0-1-per-cent.  acid. 
At  the  neutral  point  a  precipitate  is  formed  which  is  soluble  in  excess  of 
either  acid  or  alkali. 

Next  remove  B.  This  also  now  contains  alkali- albumin.  Add  to  it  a  few 
drops  of  sodium  phosphate,  colour   with  litmus,  and  neutralise  as  before. 

'  A  convenient  form  of  warm  bath  suitable  for  class  purposes  may  be  made  by 
placing  an  ordinary  tin  pot  half  full  of  water  over  a  bent  piece  of  iron  which 
acts  as  a  warm  stage  as  in  the  figure.  The  stage  is  kept  warm  by  a  small  gas 
flame.     Such  a  warm  bath  may  be  placed  between  every  two  or  three  students. 


30  ESSENTIALS   OF   CHEMICAL   PHYSIOLOGY 

Note  that  the  alkali-albumin  now  requires  more  acid  for  its  precipitation 
than  in  A,  the  acid  which  is  first  added  converting  the  sodium  phosphate  into 
acid  sodium  phosphate. 

Now  remove  C  from  the  bath.  Boil  it.  Again  there  is  no  coagulation, 
the  proteins  having  been  converted  into  acid-alhumin,  or  syntonin.  After 
cooling,  colour  with  litmus  and  neutralise  with  O'l-per-cent.  alkali.  At  the 
neutral  point  a  precipitate  is  formed,  soluble  in  excess  of  acid  or  alkali. 
(Acid-albumin  is  formed  more  slowly  than  alkali-albumin,  so  it  is  best  to 
leave  this  experiment  to  the  last.) 

2.  Take  some  gelatin  and  dissolve  it  in  hot  water.  On  cooling,  the  solu- 
tion sets  into  a  jelly  (gelatinisation). 

Take  a  dilute  solution  of  gelatin,  and  try  all  the  protein  tests  with  it 
enumerated  on  p.  27.     Carefully  note  down  your  results. 

3.  Add  a  few  drops  of  acetic  acid  to  some  saliva.  A  stringy  precipitate 
of  mucin  is  formed. 

4.  A  tendon  has  been  soaked  for  a  few  daj'S  in  lime  water.  The  fibres 
are  not  dissolved,  but  they  are  loosened  from  one  another  owing  to  the  solu- 
tion of  the  interstitial  or  ground  substance  by  the  lime  water.  Take  some 
of  the  lime-water  extract  and  add  acetic  acid.  A  precipitate  of  mucoid  is 
obtained.  The  fibres  themselves  consist  of  collagen,  which  yields  gelatin  on 
boiling.  Vitreous  humour  or  the  Whartonian  jelly  of  the  umbilical  cord  is 
much  richer  in  ground  substance  than  tendon,  and,  if  treated  in  the  same 
way,  a  much  larger  yield  of  mucoid  is  obtained. 

The  Proteins  are  the  most  important  substances  that  occur  in 
animal  and  vegetable  organisms,  and  protein  metabolism  is,  as  already 
noted,  the  most  characteristic  sign  of  life. 

They  are  highly  complex  compounds  of  carbon,  hydrogen,  oxygen, 
nitrogen,  and  sulphur  occurring  in  a  solid  viscous  condition,  or  in 
solution  in  nearly  all  parts  of  the  body.  The  different  members  of 
the  group  present,  how^ever,  great  differences  in  their  chemical  and 
physical  properties. 

The  proteins  in  the  food  form  the  source  of  the  proteins  in  the 
body  tissues,  but  the  latter  are  usually  different  in  composition  from 
the  former.  The  food  proteins  are  in  the  process  of  digestion 
broken  up  into  simpler  substances,  usually  called  cleavage  products, 
and  it  is  from  these  that  the  body  cells  reconstruct  the  proteins 
peculiar  to  themselves.  As  a  result  of  katabolic  processes  in  the 
body,  the  proteins  are  finally  again  broken  down,  carbonic  acid, 
water,  sulphuric  acid  (combined  as  sulphates),  urea,  and  creatinine 
being  the  principal  final  products  which  are  discharged  in  the  urine  and 
other  excretions.  The  intermediate  substances  between  the  proteins 
and  such  final  katabolites  as  urea  will  be  discussed  under  Urine. 

The  following  figures  will  convince  the  student  how  different  the 
proteins  are  in  elementary  composition ;  Hoppe-Seyler  many  years 
ago  gave  the  variations  in  percentage  composition  as  follows  : — 

a  H  N  3  0 

From         51-5        69         15-2        0-3         20-9 
To  5i'5         7-3         17-0        2-0        23*5 


THE   PROTEINS  31 

and  recent  research  has  since  shown  that  the  variations  are  even 
greater  than  those  just  stated. 

The  same  fact  is  brought  home  more  vividly  when  the  cleavage 
products  are  separated  and  estimated.  These  differ  both  in  kind 
and  in  amount,  but  nearly  all  of  them  are  substances  which  are 
termed  amino-acids.  Emil  Fischer,  to  whom  we  owe  so  much  of  our 
knowledge  in  this  direction,  considers  that  the  proteins  are  linkages 
of  a  greater  or  lesser  number  of  these  amino-acids,  and  there  is  great 
hope  that  in  the  future  his  work  will  result  in  an  actual  synthesis  of 
the  protein  molecule,  and  with  that  will  come  an  accurate  knowledge 
of  its  constitution. 

When  the  protein  molecule  is  broken  down  in  the  laboratory 
by  processes  similar  to  those  brought  about  by  the  digestive 
ferments  which  occur  in  the  alimentary  canal,  the  essential 
change  is  due  to  what  is  called  hydrolysis  :  that  is,  the  molecule 
unites  with  water  and  then  breaks  up  into  smaller  molecules.  The 
first  cleavage  products,  which  are  called  proteoses,  retain  many  of 
the  characters  of  the  original  protein  ;  and  the  same  is  true,  though 
to  a  less  degree,  of  the  peptones,  which  come  next  in  order  of  for- 
mation. The  peptones  in  their  turn  are  decomposed  into  short  link- 
ages of  amino-acids  which  are  called  polypeptides,  and  finally  the 
individual  amino-acids  are  obtained  separated  from  each  other. 

What  we  have  already  learnt  about  the  fatty  acids  will  help  us  in 
understanding  what  is  meant  by  an  amino-acid. 

If  we  take  acetic  acid,  which  is  one  of  the  simplest  of  the  fatty 
acids,  we  see  that  its  formula  is 

CH3.COOH. 

If  one  of  the  three  hydrogen  atoms  in  the  CH3  group  is  replaced 
by  NH2  we  get  a  substance  which  has  the  formula 

CH2.NH2.COOH. 

The  combination  NH2  which  has  stepped  in  is  called  the  amino- 
group,  and  the  new  substance  now  formed  is  called  amino-acetic  acid ; 
it  is  also  termed  glycine  or  glycocoll. 

We  may  take  another  example  from  another  fatty  acid.  Propionic 
acid  is  C2H5.COOH ;  if  we  replace  an  atom  of  hydrogen  by  the 
amino-group  as  before,  we  obtain  C2H4.NH2.COOH,  which  is  amino- 
propionic  acid  or  alanine.  Going  a  little  higher  in  the  scale,  and  taking 
caproic  acid,  OsHn^COOH,  we  obtain  from  it,  in  an  exactly  similar 
way,  CsHio.NHg.COOH,  which  is  amino-caproic  acid  or  leucine. 

All  the  three  amino-acids  mentioned— glycine,  alanine,  and  leucine 


32  ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 

— are  found  among  the  final  cleavage  products  of  most  proteins;  but 
there  are  a  good  many  more  in  addition,  for  instance  : — 

Serine  (aminO-oxypropionic  acid,  CHaOH.CHNHo.COOH) ; 

Amino-valeric  acid ; 

Amino-succinamic  acid  (asparagine)  ; 

Amino-succinic  acid  (aspartic  acid)  ; 

Amino-pyrotartaric  acid  (glutamic  acid), 
some  of  which  are  derived  from  fatty  acids  of  a  different  series  from 
those  first  enumerated. 

But  in  all  these  cases  there  is  only  one  replacement  of  an  atom 
of  hydrogen  by  NH2 ;  hence  they  are  called  monoamino-acids. 

Passing  to  the  next  stage  in  complexity,  we  come  to  another 
group  of  amino-acids  which  are  called  diamino-acids :  that  is,  fatty 
acids  in  which  two  hydrogen  atoms  are  replaced  by  NH^  groups.  Of 
these  we  may  mention  lysine,  ornithine,  arginine,  and  histidine. 

Lysine  is  diamino-caproic  acid. 

Caproic  acid  is  C5H11.GOOH. 

Mono-amino-caproic  acid,  or  leucine,  we  have  already  learnt  is 
C5H10.NH2.COOH. 

Lysine  or  diamino-caproic  acid  is  C5H,j(NH2)2COOH. 

OrnitMne  is  di-amino-valeric  acid,  and  the  following  formulae  will 
show  its  relationship  to  its  parent  fatty  acid  : — 
C4H9.COOH  is  valeric  acid. 
C4H7(NH2)2.COOH  is  diamino-valeric  acid  or  ornithine. 

Arginine  is  a  somewhat  more  complex  substance,  which  contains 
the  ornithine  radical.  It  belongs  to  the  same  group  of  substances  as 
creatine,  another  important  cleavage  product  of  the  protein  molecule. 

Creatine  is  methyl-guanidine  acetic  acid,  and  has  the  formula 

HN\    ; 

C ;  -N(CH3)0H2.COOH 

On  boiling  it  with  baryta  water,  it  takes  up  water  (H2O)  and  splits 
at  the  dotted  line  into  urea  [CO   (NH)2]  and  sarcosine,    as   shown 

below. 

H,N\ 

C=0  NH.CH3.CH2.COOH 
H.N/ 

[urea]  [surcosiiie  or  inethyl-glyciue] 

Arginine  decomposes  in  a  similar  way,  urea  being  split  off  on 
the  left,  and  ornithine  instead  of  sarcosine  on  the  right.  Arginine  is 
therefore  a  compound  of  ornithine  with  a  urea  group. 

Histidine,   the    last    member   of    this    class,  has    the    formula 


I 


THE  PEOTEINS  33 

CfjHgNsOs  is  probably  also  a  diamino-acid,  but  its  exact  constitution 
has  not  yet  been  made  out  with  certainty. 

These  substances  we  have  hitherto  described  as  acids,  but  they 
may  also  play  the  part  of  bases,  the  introduction  of  a  second  amino- 
group  into  the  fatty-acid  molecules  conferring  upon  them  basic  pro- 
perties.    The  three  substances 

Lysine       (CeHnNsOs) 
Arginine    (C6H14N4O2) 
Histidine  (OeHgNgOs) 
are  in  fact  often  called  the  hexone  bases  because  each  of  them  contains 
six  atoms  of  carbon,  as  the  above  empirical  formulae  show. 

But  there  is  still  an  important  group  of  amino-acids  to  be  con- 
sidered, and  these  are  termed  the  aromatic  amino-acids  :  that  is,  amino- 
acids  united  to  the  benzene  ring ;  and  of  these  we  shall  mention 
three  :  namely,  phenyl-alanine,  tyrosine,  and  a  nearly  related  sub- 
stance called  tryptophane. 

Phenyl-alanine  is  alanine  or  amino-propionic  acid  in  which  an 
atom  of  hydrogen  is  replaced  by  phenyl  (C6H5). 

Propionic  a.cid  has  the  formula  C2H5.COOH. 
Alanine  (amino-propionic  acid)  is  C2H4.NH2.COOH. 
Phenyl-alanine  is  C2H3.GGH5.NH2.COOH. 
The  formula  of  phenyl-alanine  may  also  be  written  another  way. 
The  graphic  formula  of  benzene  (C^H^)  is : 

H 

I 
C 

^  \ 

H— C  C— H 

I  '^^  t  II 
.     H— C  "^i  C— H 

If   the  H  placed  lowermost   in   the  above  formula  is  replaced   by 
CH2.CH.NH2.COOH  we  obtain  the  formula  of  phenyl-alanine  : — 


CH2.CHNH0COOH 

the  remainder  of  the  benzene  ring  which  is  unaltered  being  repre- 
sented, as  usual,  by  a  simple  hexagon. 

D 


34  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

Tyrosine  is  a  little  more  complicated  :  it  is  oxyphenyl-alanine  : 
that  is,  instead  of  phenyl  (CeH.^)  in  the  formula  of  phenyl-alanine  we 
have  now  oxyphenyl  (CgHj.OH).  This  gives  us  C2H3.(OoH4.0H) 
NH2.COOH  as  the  formula  for  tyrosine  written  one  way,  or 

OH 


CH2.CH.NH2.COOH 

when  written  in  the  other  way. 

Tryptophane  is  more  complex  still ;  it  is  indole-amino-propionic 
acid  :  that  is,  amino-propionic  acid  united  to  another  ringed  derivative 
called  indole.  Tryptophane  is  the  portion  of  the  protein  molecule 
which  is  the  parent  substance  of  two  evil-smelling  products  of  protein 

decomposition  called  indole  CGHiZ-^r-rr" 

and  scatole  or  methyl  indole.  Tryptophane  is  also  the  radical  in  the 
protein  molecule  which  is  responsible  for  the  colour  test  called  the 
Adamkiewicz  reaction. 

We  may  sum  up  what  we  have  learnt  up  to  this  point  by  enume- 
rating the  principal  members  of  these  three  groups  of  amino- 
acids :  — 

1.  The  mono-amino-acids :  glycine,  alanine,  leucine,  amino- 
valeric  acid,  asparagine,  aspartic  acid,  glutamic  acid. 

2.  The  di-amino -acids  :  lysine,  ornithine,  arginine,  and  histidine. 

3.  The  ringed  amino -acids :  phenyl-alanine,  tyrosine,  and 
tryptophane. 

But  this  does  not  bring  us  to  the  end  of  the  list  of  the  cleavage 
products  of  proteins,  for  we  have  still  left  several  other  groups  most 
of  which  are  still  more  complex,  and  which  we  will  therefore  be 
content  with  merely  mentioning  :  namely — 

4.  Pyrimidine  bases  such  as  uracil,  thymine  and  cytosine. 

5.  Pyrrolidine  derivatives. 

6.  Cystine,  a  complex  amino- acid  in  which  sulphur  is  present,  and 
in  which  the  greater  part  of  the  sulphur  of  the  protein  molecule  is 
combined. 

7.  Ammonia. 

Our  list  now  represents  the  principal  groups  of  chemical  nuclei 
united  together  in  the  protein  molecule,  and  its  length  makes  one 


THE   PROTEINS 


35 


realise  the  complicated  nature  of  that  molecule  and  the  difficulties 
which  beset  its  investigation. 

The  workers  in  Fischer's  laboratory  are  steadily  working  through 
the  various  known  proteins,  taking  them  to  pieces,  and  identifying 
and  estimating  the  fragments.  I  do  not  intend  to  burden  the  readers 
of  this  book  with  anything  more  than  a  sample  of  their  results,  and 
will  therefore  only  give  in  a  brief  table  the  results  obtained  with 
some  of  the  cleavage  products  of  a  few  proteins.  The  numbers 
given  are  percentages. 


Edes. 

irl 

Egg-al- 
bumin 

Serum 
globu- 
lin 

Casei- 
nogen 

Gelatin 

Keratin 
from  horse- 
hair 

tin,  a 
globu- 
lin from 
hemp 

Zein 
from 
maize 

Glia. 

din 

from 

wheat 

1  Glycine     . 

0 

0 

8-5 

0 

16-5 

seed 

0-9 

0-8 

3.8 

+ 

'  Leucine     . 

20-0 

6-1 

18-7 

10-5 

2-1 

7-1 

15-5 

11-2 

6-0 

Glutamic  acid  . 

7-7 

8-0 

8-5 

11-0 

0-8 

3-7 

17-2 

11-8 

31-5 

Tyrosine   . 

2-1 

1-1 

2-5 

4-5 

0 

3-2 

2-1 

10-1 

2-4 

Arginine    . 

4-8 

7-6 

11.7 

1-8 

2-75 

Tryptophane     . 

+ 

-f 

+ 

1-5 

0 

more 

-f 

!  Cystine  . 

2-3 

0-2 

0*7 

0-06 

than  10 

0-2 

Such  numbers  of  course  are  not  to  be  committed  to  memory,  but 
they  are  sufficient  to  convey  to  the  reader  the  differences  between  the 
proteins.  There  are  several  blanks  left  on  account  of  no  accurate 
estimations  having  yet  been  made.  Where  the  sign  -f  occurs,  the 
substance  in  question  has  been  proved  to  be  present,  but  not  yet 
determined  quantitatively.  Among  the  more  striking  points  brought 
out  are  : — 

1.  The  absence  of- glycine  from  albumins. 

2.  The  high  percentage  of  glycine  in  gelatin. 

3.  The  absence  of  tyrosine  and  tryptophane  in  gelatin. 

4.  The  high  percentage  of  the  sulphur  containing  substance 
(cystin)  in  keratin. 

5.  The  high  percentage  of  glutamic  acid  in  vegetable  proteins. 
Emil  Fischer  in  his  work  has  sought  to  make  such  a  list  complete, 

and  month  by  month  the  details  are  being  filled  in.  He  has  next 
tried  to  discover  the  way  in  which  the  amino-acids  are  linked  to- 
gether into  groups ;  and  the  culmination  of  his  work  will  be  the 
discovery  of  the  way  in  which  such  groups  are  linked  together  to 
form  the  protein  molecule.  The  last  stage  he  has  not  yet  reached, 
but  it  will  be  interesting  to  see  what  progress  he  has  -made  in 
ascertaining  how  the  amino-acids  are  linked  together  into  groups. 

D  2 


36  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

These  groups  he  terms  peptides  or  polypeptides :  many  of  these 
have  been  made  synthetically  in  his  laboratory,  and  so  the  synthesis 
of  the  protein  molecule  is  foreshadowed. 

We  may  take  as  our  examples  of  these  peptides  some  of  the 
simplest,   and   may   write   the  formulae   of    a   few    amino-acids   as 

follows : — 

NH2.CH2.COOH glycine 

NH2.CH4.COOH alanine 

NH2.C5H10.COOH leucine 

or  in  general  terms 

HNH.E.COOH 

Two  amino-acids  are  linked  together  as  shown  in  the  following 
formula  : — 

HNH.K.CO  OH.H  NH.R.COOH 


What  happens  is  that  thehydroxyl  (OH)  of  the  carboxyl  (COOH) 
group  of  one  acid  unites  with  one  atom  of  the  hydrogen  of  the  next 
amino  (HNH)  group,  and  water  is  thus  formed,  as  shown  within  the 
dotted  lines :  this  is  eliminated  and  the  rest  of  the  chain  closes  up. 
In  this  way  we  get  a  dipeptide.  The  names  glycyl,  alanyl,  leucyl, 
&c.  are  given  by  Fischer  to  the  NH2.R.CO  groups  which  replace 
the  hydrogen  of  the  next  NH2  group.  Thus  glycyl-glycine, 
glycyl-leucine,  leucyl-alanine,  alanyl-leucine,  and  numerous 
other  combinations  and  permutations  are  obtained.  If  the  same 
operation  is  repeated  we  obtain  tripeptides  (leucyl- glycyl-alanine. 
alanyl-leucyl-tyrosine  &c.)  ;  then  come  the  tetrapeptides  and  so  on. 
In  the  end,  by  coupling  the  chains  sufficiently  often  and  in  appro- 
priate order,  Fischer  has  already  obtained  substances  which  give  some 
of  the  reactions  of  peptones. 

TESTS  FOR  PROTEINS 

Solubilities. — The  proteins  as  a  class  are  insoluble  in  alcohol  and 
in  ether.  Some  are  soluble  in  water,  others  insoluble.  Many  of  the 
latter  are  soluble  in  weak  saline  solutions.  Some  are  soluble,  others 
insoluble  in  concentrated  saUne  solutions.  It  is  very  largely  on 
these  varying  solubilities  that  proteins  are  separated  into  classes, 
and  from  each  other. 

When,  however,  one  speaks  of  the  solution  of  a  protein,  the  kind 
of  solution  obtained  is  usually  what  physical  chemists  call  colloidal 
solution :  the  condition  here  is  something  intermediate  between  true 
solution  and  a  suspension.     Many  of  the  properties  of  proteins,  such 


THE   PEOTEINS 


37 


as  the  tendency  to  coagulate  or  solidify  and  the  readiness  with 
which  they  are  '  salted  out,'  are  shared  in  common  with  other  colloids 
some  of  which  aie  of  inorganic  nature. 

All  proteins  are  soluble  with  the  aid  of  heat  in  concentrated 
mineral  acids  and  alkalis.  Such  treatment,  however,  decomposes  as 
well  as  dissolves  the  protein.  Proteins  are  also  soluble  in  gastric  and 
pancreatic  juices ;  but  there,  again,  they  undergo  a  change,  being 
converted  by  hydrolysis  into  proteins  of  smaller  molecular 
weight  called  peptones.  The  intermediate  substances  formed  in  this 
process  are  called  proteoses.  Commercial  peptone  contains  a  mixture 
of  proteoses  and  true  peptone. 

Heat  Coagulation. — Many  of  the  proteins  which  are  soluble  in 
water  or  saline  solution  are   rendered  insoluble  when  those  solutions 


Fig.  7.--Dialyser.  The  lower  opening  of  the  bell  jar 
suspended  in  water  is  tightly  covered  with  parchment 
paper.  The  fluid  to  be  dialysed  is  placed  within  this 
vessel;  the  crystalloids  pass  out  into  the  distilled 
water  outside  through  the  parchment  paper. 


Fig,  8. — In  this  form  of  dialyser 
the  substance  to  be  dialysed 
is  placed  within  the  piece 
of  tubing  suspended  in  the 
larger  vessel  of  water.  The 
tubing  is  made  of  parchment 
paper. 


are  heated.  This  is  true  for  most  of  the  proteins  that  occur  in  nature. 
The  solidifying  of  white  of  egg  when  heated  is  a  familiar  instance  of 
this.  The  temperature  of  heat  coagulation  differs  in  different  pro- 
teins :  thus  myosinogen  and  fibrinogen  coagulate  at  about  56°  C.  ; 
serum  albumin  and  serum  globulin  at  about  75°  C. 

The  proteins  which  are  coagulated  by  heating  their  solution  come 
for  the  most  part  into  two  classes — the  alhimiins  and  the  globulins. 
The  full  distinction  between  these  we  shall  see  immediately.  We 
may,  however,  state  here  that  the  albumins  are  soluble  in  distilled 


38  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

water  :  the  true  globulins  are  not,  but  require  salts  to  hold  them  in 
solution. 

Indiffusibility. — The  proteins  (peptones  excepted)  belong  to  the 
class  of  substances  called  colloids  by  Thomas  Graham  :  they  pass 
with  difficulty,  or  not  at  all,  through  animal  membranes.  In  the 
construction  of  dialysers,  vegetable  parchment  is  very  largely  used 
(see  figs.  7  and  8). 

Proteins  may  thus  be  separated  from  diffusible  (crystalloid)  sub- 
stances like  salts,  but  the  process  is  a  somewhat  tedious  one.  If 
some  serum  or  white  of  egg  is  placed  in  a  dialyser,  and  distilled 
water  outside,  the  greater  amount  of  the  salts  passes  into  the  water 
through  the  membrane  ;  the  two  proteins,  albumin  and  globulin, 
remain  inside.  The  globulin  is,  however,  precipitated,  as  the  salts 
which  previously  kept  it  in  solution  have  been  removed. 

The  terms  '  diffusion '  and  '  dialysis '  should  be  distinguished  from  each 
other. 

If  water  is  carefully  poured  on  the  surface  of  a  solution  of  any  substance, 
this  substance  gradually  spreads  through  the  water,  and  the  composition  of 
the  mixture  becomes  uniform  in  time.  The  time  occupied  is  short  for 
substances  like  sodium  chloride,  and  long  for  substances  like  albimiin.  The 
phenomenon  is  called  diffusion.  If  the  solutions  are  separated  by  a  mem- 
brane the  term  '  dialysis '  is  employed.  The  word  osmosis  is  properly  restricted 
to  the  passage  of  water  through  membranes,  and  can  be  best  studied  when 
semi-permeable  membranes  are  employed.  See  more  fully  article  Osmosis  in 
Appendix. 

Crystallisation. — Haemoglobin,  the  red  pigment  of  the  blood,  is  a 
protein  substance,  and  is  crystallisable  (for  further  details,  see  The 
Blood).  Like  other  proteins,  it  has  an  enormously  large  molecule  ; 
though  crystalline,  it  is  not  crystalloid  in  Graham's  sense  of 
that  term,  although  it  probably  forms  a  true  solution  with  water. 
Blood  pigment  is  not  the  only  crystallisable  protein.  Long  ago 
crystals  of  protein  (globulin  or  vitellin)  were  observed  in  the  aleurone 
grains  of  many  seeds,  and  in  the  similar  protein  occurring  in 
the  egg-yolk  of  some  fishes  and  amphibians.  By  appropriate 
methods  these  have  been  separated  and  recrystallised.  Further, 
egg-albumin  itself  has  been  crystallised.  If  a  solution  of  white  of  egg 
is  diluted  with  half  its  volume  of  saturated  solution  of  ammonium 
sulphate,  the  globulin  present  is  precipitated  and  is  removed  by 
filtration.  The  filtrate  is  now  allowed  to  remain  some  days  at  the 
temperature  of  the  air,  and  as  it  becomes  more  concentrated  from 
evaporation,  minute  spheroidal  globules  and  finally  minute  needles, 
either  aggregated  or  separate,  make  their  appearance  (Hofmeister). 
Crystallisation  is  much  more  rapid  and  perfect  if  a  little  acetic  or 


THE   PROTEINS  39 

sulphuric  acid  is  added  (Hopkins).  Serum  albumin  (from  come 
animals)  has  also  been  similarly  crystallised  (Giirber). 

Action  on  Polarised  Light. — All  the  proteins  are  levo-rotatory, 
but  the  amount  of  rotation  the}  produce  varies  with  the  kind  of 
protein.  See  Appendix.  Several  of  the  compound  proteins  (for 
instance,  haemoglobin  and  nucleo-proteins)  are  dextro-rotatory, 
though  their  protein  components  are  levo-rotatory  (Gam gee). 

Colour  Reactions. — The  principal  colour  reactions  have  been 
already  described  in  the  heading  of  this  lesson. 

(1)  The  xantho-proteic  reaction  depends  on  the  conversion  of  the 
aromatic  group  of  the  protein  molecule  into  nitro-derivatives. 

(2)  Millon's  reaction  is  due  to  the  presence  of  the  tyrosine  group, 
and  is  given  by  all  benzene  derivatives  which  contain  a  hydroxyl 
group  (OH)  replacing  hydrogen. 

(3)  The  formaldehyde  reaction  (and  the  Adamkiewicz  reaction, 
which  is  probably  the  same  thing)  is  due  to  the  presence  of  the  trypto- 
phan radical  (indole-amino-propionic  acid). 

The  presence,  absence,  or  intensity  of  these  colour  tests  in  various 
proteins  depends  respectively  on  the  presence,  absence,  or  amount  of 
the  groups  to  which  they  are  due. 

(4)  In  the  copper  sulphate  test  the  proteoses  and  peptones 
behave  differently  from  the  native  proteins  ;  the  latter  give  a  violet 
and  the  former  a  rose-red  colour,  which  is  called  the  biuret  reaction, 
because  the  same  tint  is  also  given  by  the  substance  called  biuret.^ 
The  name  does  not  imply  that  biuret  is  present  in  protein,  but  both 
biuret  and  protein  give  the  reaction  because  they  possess  the  same 
group  or  groups  which  are  probably  two  GONH2  groups  linked  either 
to  a  carbon  atom,  or  to  a  nitrogen  a,tom,  or  directly  to  one  another 
(Schiff). 

Precipitants  of  Proteids. — Proteids  are  precipitated  by  a  large 
number  of  reagents ;  the  peptones  and  proteoses  are  exceptions  in 
many  cases,  and  will  be  considered  separately  afterwards  (see 
Lesson  VH.). 

Solutions  of  the  proteins  are  precipitated  by — 

1.  Strong  acids,  like  nitric  acid. 

2.  Picric  acid. 

3.  Acetic  acid  and  potassium  ferrocyanide. 

4.  Acetic  acid  and  excess  of  neutral  salts  like  sodium  sulphate. 

'  Biuret  is  obtained  by  heating  solid  urea ;  ammonia  is  given  off  and  leaves 
biuret  thus : — 

2C0N,H,=     CANgHs     +NH3 

[urea]  [biuret]      [ammouia] 


40  ESSENTIALS  OF  CHEMICAL  PHYSIOLOaY 

5.  Salts  of  the  heavy  metals,  like  copper  sulphate,  mercuric 
chloride,  lead  acetate,  silver  nitrate,  &c. 

6.  Tannin. 

7.  Alcohol. 

8.  Saturation  with  certain  neutral  salts,  such  as  ammonium 
sulphate. 

It  is  necessary  that  the  words  coagulation  and  precipitation  should, 
in  connection  with  the  proteins,  be  carefully  distinguished.  The 
term  coagulation  is  used  when  an  insoluble  protein  (coagulated 
protein)  is  formed  from  a  soluble  one.     This  may  occur — 

1.  When  the  protein  is  heated — heat  coagulation. 

2.  Under  the  influence  of  a  ferment ;  for  instance,  when  a  curd  is 
formed  in  milk  by  rennet  or  a  clot  in  shed  blood  by  the  fibrin  ferment 
— ferment  coagulation. 

3.  When  an  insoluble  precipitate  is  produced  by  an  addition  of 
certain  reagents  (nitric  acid,  picric  acid,  tannin,  &c.). 

There  are,  however,  other  precipitants  of  proteins  in  which  the 
precipitate  formed  is  readily  soluble  in  suitable  reagents,  like  saline 
solution,  and  the  protein  continues  to  show  its  typical  reactions. 
This  precipitation  is  not  coagulation.  Such  a  precipitate  is  produced 
by  saturation  with  ammonium  sulphate.  Certain  proteins,  called 
globulins,  are  more  readily  precipitated  by  such  means  than  others. 
Thus,  serum  globulin  is  precipitated  by  half-saturation  with  ammonium 
sulphate.  Full  saturation  with  ammoniun  sulphate  precipitates  all 
proteins  but  peptone.  The  globulins  are  precipitated  by  certain  salts 
like  sodium  chloride  and  magnesium  sulphate,  which  do  not  precipi- 
tate the  albumins.  The  precipitation  of  proteins  by  salts  in  this  way 
is  conveniently  termed  '  salting  out.' 

The  precipitate  produced  by  alcohol  is  peculiar  in  that  after 
a  time  it  becomes  a  coagulum.  Protein  freshly  precipitated  by 
alcohol  is  readily  soluble  in  water  or  saline  media  ;  but  after  it  has 
been  allowed  to  stand  some  weeks  under  alcohol  it  becomes  more 
and  more  insoluble.  Albumins  and  globulins  are  most  readily  ren- 
dered insoluble  by  this  method  ;  proteoses  and  peptones  are  appa- 
rently never  rendered  insoluble  by  the  action  of  alcohol.  This  fact 
is  of  value  in  the  separation  of  these  proteins  from  others. 


THE   PROTEINS  41 

CLASSIFICATION   OF  PROTEINS 

The  knowledge  of  the  chemistry  of  the  proteins  which  is  slowly 
progressing  under  Emil  Fischer's  leadership  will  no  doubt  in  time 
enable  us  to  give  a  classification  of  the  substances  on  a  strictly 
chemical  basis.  But  until  that  time  arrives  we  must  be  content  very 
largely  with  the  artificial  classification  (on  the  basis  of  solubility  and 
so  forth)  which  has  hitherto  prevailed.  The  following  classification 
must  therefore  be  regarded  as  a  provisional  one,  which,  while  it  retains 
the  old  familiar  names  as  far  as  possible,  yet  attempts  also  to 
incorporate  some  of  the  new  ideas.  The  classes  of  proteins,  then, 
beginning  with  the  simplest,  are  as  follow  : — 

1.  Protamines. 

2.  Histones. 

3.  Albumins. 

4.  Globulins. 

5.  Sclero-proteins. 

6.  Phospho-proteins. 

7.  Conjugated  proteins. 

(a)  Gluco-proteins. 

(b)  Nucleo-proteins. 

(c)  Chromo-proteins. 
We  shall  take  these  classes  one  by  one. 

1.  The  Protamines 

These  substances  are  obtainable  from  the  heads  of  the  spermatozoa 
of  certain  fishes,  where  they  occur  in  combination  with  nuclein. 
Kossel's  view  that  they  are  the  simplest  proteins  in  nature  has  met 
with  general  acceptance,  and  they  give  such  typical  protein  reactions 
as  the  copper  sulphate  test  (Eose's  or  Piotrowski's  reaction).  On 
hydrolytic  decomposition  they  first  yield  substances  of  smaller 
molecular  weight  analogous  to  the  peptones  which  are  called  protones, 
and  then  they  split  up  into  amino-acids.  The  number  of  resulting 
amino-acids  is  small  as  compared  with  other  proteins  ;  hence  the 
hypothesis  that  they  are  simple  proteins  is  confirmed.  Notable 
among  their  decomposition  products  are  the  diamino-acids  or  hexone 
bases,  which  have  the  following  names  and  formulae  : — 

Lysine       (OeHnNaOa) 

Arginine    (C6H.,N402) 

Histidine  (CgHgNaOs) 
The  protamines  differ  in  their  composition  according  to  their  source, 
and  yield  these  products  in  different  proportions. 


42  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

Salmine  (from  the  salmon  roe)  and  clupeine  (from  the  herring  roe) 
appear  to  be  identical,  and  have  the  empirical  formula  C30H57N17O6  : 
its  principal  decomposition  products  are  arginine,  amino- valeric  acid, 
and  a  small  quantity  of  an  unknown  residue.  Sturinc  (from  the 
sturgeon)  yields  the  same  products  v^rith  lysine  and  histidine  in 
addition.  With  one  exception,  the  protamines  yield  .no  aromatic 
amino-acids,  of  w^hich  tyrosine  is  a  familiar  instance ;  the  exception 
is  cyclopterine  (from  Cycloptertos  lumpus) ;  this  substance  is  thus  an 
important  chemical  link  between  the  other  protamines,  and  the  more 
complex  members  of  the  protein  family. 

2.  The  Histones 

These  are  substances  which  have  been  separated  from  blood 
corpuscles  ;  glohin,  the  protein  constituent  of  heemoglobin,  is  a  well- 
marked  instance.  They  yield  a  larger  number  of  amino-compounds 
than  do  the  protamines.  They  are  coagulable  by  heat,  soluble  in 
dilute  acids,  and  precipitable  from  such  solutions  by  ammonia.  The 
precipitability  by  ammonia  is  a  property  possessed  by  no  other 
protein  group. 

3.  The  Albumins 

These  are  typical  proteins,  and  yield  the  majority  of  the  cleavage 
products  enumerated  on  pp.  31-35. 

They  enter  into  colloidal  solution  in  water  in  dilute  saline  solutions, 
and  in  saturated  solutions  of  sodium  chloride  and  magnesium 
sulphate.  They  are,  however,  precipitated  by  saturating  their 
solutions  with  ammonium  sulphate.  Their  solutions  are  coagulated 
by  heat  usually  at  70°-73°  C.  Serum  albumin,  egg-albumin,  and 
lact-albumin  are  instances. 

4.  The  Globulins 

The  globulins  give  the  same  general  tests  as  the  albumins :  they 
are  coagulated  by  heat,  but  differ  from  the  albumins  mainly  in  their 
solubiHties.  This  difference  in  solubility  may  be  stated  in  tabular  form 
as  follows : — 


Reagent  Albumin 


Globulin 


Water I  soluble  insoluble 

Dilute  saline  solution        .         .         .  soluble  |  soluble 

Saturated    solution    of    magnesium 

sulphate  or  sodium  chloride  .  ^  soluble  insoluble 

Half- saturated  solution  of  ammo- 
nium sulphate        .... 

Saturated    solution    of    ammonium 

sulphate i  insoluble  insoluble 


soluble  insoluble 


THE   PROTEINS  43 

In  general  terms  globulins  are  more  readily  salted  out  than 
albumins  ;  they  may  therefore  be  precipitated  and  thus  separated 
from  the  albumins  by  saturation  with  such  salts  as  sodium  chloride, 
or,  better,  magnesium  sulphate,  or  by  half  saturation  with  ammo- 
nium sulphate. 

The  typical  globulins  are  also  insoluble  in  water,  and  so  may  be 
precipitated  by  removing  the  salt  which  keeps  them  in  solution. 
This  may  be  accomplished  by  dialysis  (see  p.  38). 

Their  temperature  of  heat  coagulation  varies  considerably.  The 
following  are  the  commoner  globulins : — fibrinogen  and  serum 
globulin  in  blood ;  egg-globulin  in  white  of  egg,  myosinogen  in 
muscle,  and  crystallin  in  the  crystalline  lens.  We  must  also  include 
under  the  same  heading  certain  proteins  which  are  the  result  of 
ferment  coagulation  on  globulins,  such  as  fibrin  (see  Blood)  and 
myosin  (see  Muscle). 

The  most  striking  and  real  distinction  between  globulins  and 
albumins  is  that  the  latter  on  hydrolysis  yield  no  glycine,  whereas 
the  globulins  do. 

5.  The  Sclero-proteins 

These  substances  form  a  heterogeneous  group  of  substances, 
which  are  frequently  termed  albu7ninoids.  The  prefix  sclero-  indicates 
the  skeletal  origin  and  often  insoluble  nature  of  the  members  of  the 
group.      The  principal  proteins  under  this  head  are  the  following  : — 

1.  Collagen,  the  substance  of  which  the  white  fibres  of  connective 
tissue  are  composed.  Some  observers  regard  it  as  the  anhydride  of 
gelatin. 

2.  Ossein. — This  is  the  same  substance  derived  from  bone.^ 

3.  Gelatin. — This  substance  is  produced  by  boiling  collagen  with 
water.  It  possesses  the  peculiar  property  of  setting  into  a  jelly  when 
a  solution  made  with  hot  water  cools.  On  digestion  it  is  like  ordinary 
proteins   converted     into   peptone-like    substances    and    is    readily 

'  In  round  numbers  the  solid  matter  in  bone  contains  two  thirds  inorganic  or 
earthy  matter,  and  one  third  organic  or  animal  matter.  The  inorganic  constituents 
are  calcium  phosphate  (84  per  cent,  of  the  ash),  calcium  carbonate  (13  per  cent.), 
and  smaller  quantities  of  calcium  chloride,  calcium  fluoride,  and  magnesium 
phosphate.  The  organic  constituents  are  ossein  (this  is  the  most  abundant  , 
elastin  from  the  membranes  lining  the  Haversian  canals,  lacunae,  and  canaliculi, 
and  other  proteins  and  nuclein  from  the  bone  corpuscles.  There  is  also  a  small 
quantity  of  fat  even  after  removal  of  all  the  marrow.  Dentine  is  like  bone  chemi- 
cally, but  the  proportion  of  earthy  matter  is  rather  greater.  E?iamel  is  the  hardest 
tissue  in  the  body ;  the  mineral  matter  is  like  that  found  in  bone  and  dentine  ; 
but  the  organic  matter  is  so  small  in  quantity  as  to  be  practically  non-existent 
(Tomes).     Enamel  is  epiblastic,  not  mesoblastic,  like  bone  and  dentine. 


44  ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 

absorbed.  Though  it  will  replace  in  diet  a  certain  quantity  of  such 
proteins  and  thus  acts  as  a  *  protein-sparing '  food,  it  cannot 
altogether  take  their  place  as  a  food.  Animals  whose  sole  nitro- 
genous food  is  gelatin  waste  rapidly.  The  reason  for  this  is  that 
gelatin  contains  neither  the  tyrosine  nor  the  tryptophane  radicals,  and 
so  it  gives  neither  Millon's  nor  the  Adamkiewicz  reaction.  Animals 
which  receive  in  their  food  gelatin  to  which  tyrosine  and  tryptophane 
are  added  thrive  well.i 

4.  Chondrin. — This  is  the  name  given  to  the  mixture  of  gelatin 
and  mucoid  which  is  obtained  by  boiling  cartilage. 

5.  Elastin. — This  is  the  substance  of  which  the  yellow  or  elastic 
fibres  of  connective  tissue  are  composed.  It  is  a  very  insoluble 
material.  The  sarcolemma  of  muscular  fibres  and  certain  basement 
membranes  are  very  similar. 

6.  Keratin,  or  horny  material,  is  the  substance  found  in  the 
surface  layers  of  the  epidermis,  in  hairs,  nails,  hoofs,  and  horns.  It 
is  very  insoluble,  and  chiefly  differs  from  most  proteins  in  its  high 
percentage  of  sulphur.  A  similar  substance,  called  neurokeratin, 
is  found  in  neuroglia  and  nerve  fibres.  In  this  connection  it  is 
interesting  to  note  that  the  epidermis  and  the  nervous  system 
are  both  formed  from  the  same  layer  of  the  embryo — the  epiblast. 

6.  The  Phospho-proteins 

Vitellin  (from  egg-yolk),  caseinogen,  the  principal  protein  of  milk 
and  casein,  the  result  of  the  action  of  the  rennet-ferment  on 
caseinogen  (see  Milk),  are  the  principal  members  of  this  group.  Among 
their  decomposition  products  is  a  considerable  quantity  of  phosphoric 
acid.  They  have  been  frequently  confused  with  the  nucleo-proteins 
we  shall  be  studying  immediately,  and  the  prefix  nucleo-  so  often 
applied  to  them  is  entirely  misleading,  since  they  do  not  yield  the 
products  (purine  bases)  which  are  characteristic  of  nucleo-compounds. 

7.  The  Conjugated  Proteins 

These  very  complex  substances  are  compounds  in  which  the 
protein  molecule  is  united  to  other  organic  materials,  which  are,  as 
a  rule,  also  of  complex  nature.  This  second  constituent  of  the 
compound  is  usually  termed  a  prosthetic  group.  They  may  be 
divided  into  the  following  sub-classes. 

i.  Chromo-proteins. — These  are  compounds  of  proteins  with  a 
pigment,  which  usually  contains  iron.  They  are  typified  by 
haemoglobin  and  its  allies,  which  will  be  fully  considered  under  Blood 


THE   PKOTEINS  45 

ii.  Gluco-proteins. — These  are  compounds  of  protein  with  a  carbo- 
hydrate group.     This  class  includes  the  mucins  and  the  mucoids. 

The  mucins  are  widely  distributed  and  may  occur  in  epithelial 
cells,  or  be  shed  out  by  these  cells  (mucus,  mucous  glands,  goblet 
cells).  The  mucin  obtained  from  different  sources  varies  in  com- 
position and  reactions,  but  they  all  agree  in  the  following  points : — 

(a)  Physical  character.     Viscid  and  tenacious. 

(b)  They  are  soluble  in  dilute  alkalis,  such  as  lime  water,  and  are 
precipitable  from  solution  by  acetic  acid. 

The  mucoids  generally  resemble  the  mucins,  but  differ  from  them 
in  minor  details.  The  term  is  applied  to  the  mucin-like  substances 
which  form  the  chief  constituent  of  the  ground  substance  of  con- 
nective tissues  (tendo-mucoid,  chondro-mucoid,  &c.).  Another,  ovo- 
mucoid is  found  in  white  of  egg,  and  others  (pseudo- mucin  and 
para-mucin)  are  occasionally  found  in  dropsical  effusions,  and  in  the 
fluid  of  ovarian  cysts. 

It  is  probable  that  the  differences  between  the  mucins  and  the 
mucoids  are  due  either  to  the  nature  of  the  carbohydrate  group  or, 
more  probably,  to  the  nature  of  the  protein  to  which  it  is  united. 
The  carbohydrate  substance  in  the  majority  of  cases  is  not  sugar, 
but  a  nitrogenous  substance  which  has  a  similar  reducing  power  to 
sugar,  and  which  is  called  glucosamine  (CgH,  1O5NH2) .'  that  is,  glucose 
in  which  HO  is  replaced  by  NHg. 

Pavy  and  others  have  shown  that  a  small  quantity  of  the  same 
carbohydrate  derivative  can  be  split  off  from  various  other  proteins 
which  we  have  already  placed  among  the 
albumins  and  globulins.  It  is,  however, 
probable  that  this  must  not  be  considered 
a  prosthetic  group,  but  irS  more  intimately 
united  within  the  protein  molecule. 

iii.  Nucleo-proteins.  These  are  com- 
pounds of  protein  with  a  complex  organic 
acid  called  nucleic  acid  which  contains 
phosphorus.  They  are  found  both  in 
the  nuclei  and  cell-protoplasm  of  cells.  piasm  iompSed  of%on|i'op]iasm 
In  physical  characters  they  often  simu-  fatraSff  n'?t^vvork'o?X^l^- 

lofn  mnm'n  *^°  °''  ^"cleiu ;  and  «',  nucleolus 

late  mucin.  (ScMfer.) 

Nuclein  is   the    name   given   to   the 
chief  constituent  of  cell-nuclei.     It  is  identical  with  the  chromatin  of 
histologists  (see  fig.  9). 

On  decomposition  it 'yields  an  organic  acid  called  nucleic  acid 
together  with    a  variable  but  usually  small  amount  of- protein.     It 


46  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

contains  a  high  percentage  (10-11)  of  phosphorus.  The  nuclein 
obtained  from  the  nuclei  or  heads  of  the  spermatozoa  consists  of 
nucleic  acid  without  any  protein  admixture.  In  fishes'  spermatozoa, 
however,  there  is  an  exception  to  this  rule,  for  there  it  is,  as  we  have 
ah'eady  seen,  united  to  protamine. 

The  nucleo-proteins  of  cell  protoplasm  are  compounds  of  nucleic 
acid  with  a  much  larger  quantity  of  protein,  so  that  they  usually 
contain  only  1  per  cent,  or  less  of  phosphorus.  Some  also  contain 
iron,  and  it  is  probable  that  the  normal  supply  of  iron  to  the  body  is 
contained  in  the  nucleo-proteins  or  hcematogens  (Bunge)  of  plant  or 
animal  cells. 

Nucleo-proteins  may  be  prepared  from  cellular  structures  like 
thymus,  testis,  kidney,  &c.,  by  two  principal  methods  : — 

1.  Wooldridges  method.  The  organ  is  minced  and  soaked  in 
water  for  twenty-four  hours.  Dilute  acetic  acid  added  to  the  aqueous 
extract  precipitates  the  nucleo-protein. 

2.  Sodimn  chloride  method.  The  minced  organ  is  ground  up  in  a 
mortar  with  solid  sodium  chloride;  the  resulting  viscous  mass  is 
poured  into  excess  of  water,  and  the  nucleo-protein  rises  in  strings  to 
the  top  of  the  water. 

The  solvent  usually  employed  for  a  nucleo-protein,  by  whichever 
method  it  is  prepared,  is  a  1-per-cent.  solution  of  sodium  carbonate. 
The  relationship  of  nucleo-proteins  to  the  coagulation  of  the  blood  is 
described  under  that  heading. 

Nucleic  acid  on  decomposition  yields  phosphoric  acid,  various 
bases  of  the  xanthine  group,  and  bases  also  of  the  pyrimidine  group 
(cytosine,  uracil,  &c.).  In  some  cases  a  carbohydrate  radical  is  also 
obtained ;  thus  a  pentose  is  obtained  from  the  nucleic  acid  of  the 
pancreas,  the  liver,  and  yeast  cells.  There  appear  to  be  several 
nucleic  acids,  which  vary  in  the  relative  amount  they  yield  of  their 
decomposition  products,  especially  of  the  members  of  the  xanthine 
family,  which  are  sometimes  called  alloxuric,  or,  more  usually,  _2?2mw-e 
bases.  The  purine  bases  are  closely  allied  chemically  to  uric  acid, 
and  we  shall  have  to  study  them  again  in  relation  to  that  substance. 
The  following  diagrammatic  way  of  representing  the  decomposition 
of  nucleo-protein  will  assist  the  student  in  remembering  the  relation- 
ships of  these  substances  : — 


THE  PROTEINS 


47 


NUCLEO-PROTEIN 

subjected  to  gastric  digestion  yields 


Protein  converted  into 
peptone,  which  goes 
into  solution. 


Protein  converted  into 
acid  albumin  in  solu- 
tion. 


Nuclein,  which  remains  as  an  insoluble 
residue.  If  this  is  dissolved  in  alkali 
and  then  hydrochloric  acid  added  it 
yields 

I 

I.  . 

A  precipitate  consisting  of  nucleic  acid. 
If  this  is  heated  in  a  sealed  tube  with 
hydrochloric  acid,  it  yields  a  number 
of  substances.  But  the  best  known 
and  constant  products  of  its  decom- 
position are 


Phosphoric  acid. 


I 

Purine  bases,  viz. 
Adenine 
Hypoxanthine 
Guanine 
Xanthine. 


I 

Pyrimidine 
Uracil 

Cytosine 
Thymine. 


,  VIZ. 


Protein-hydrolysis 

When  protein  material  is  subjected  to  hydrolysis,  as  it  is  when 
heated  with  mineral  acid,  or  superheated  steam,  or  to  the  action  of 
such  ferments  as  pepsin  or  trypsin  in  the  alimentary  canal,  it  is 
finally  resolved  into  the  numerous  amino-acids  of  which  it  is  built. 
But  before  this  ultimate  stage  is  reached,  it  is  split  into  substances  of 
progressively  diminishing  molecular  size,  which  still  retain  many  of 
the  protein  characters.  These  may  be  classified  in  order  of  formation 
as  follows : — 

1.  Infra-proteins. 

2.  Proteoses. 

3.  Peptones. 

4.  Polypeptides. 

The  polypeptides  are  linkages  of  two  or  more  amino-acids  as  already 
explained.  They  do  not  give  the  biuret  reaction.  Although  most 
of  the  polypeptides  at  present  known  are  products  of  laboratory 
synthesis,  some  have  been  definitely  separated  from  the  digestion  of 


48  ESSENTIALS   OF  CHEMICAL   PHYSIOLOGY 

proteins,  and  so  they  must  appear  in  our  classification.  The  proteoses 
and  peptones  give  the  biuret  reaction  ;  the  peptones,  however,  cannot 
be  salted  out  of  solution  like  the  proteoses ;  their  molecules  are 
smaller  than  those  of  the  proteoses.  We  shall  study  them  more 
fully  under  digestion.  Tt  is,  however,  necessary  to  add  here  a  brief 
description  of  the  infra-proteins,  since  some  of  the  practical  exercises 
at  the  head  of  this  lesson  deal. with  them. 

They  are  obtained  as  the  first  stage  of  hydrolysis,  and  also  by 
the  action  of  dilute  acids  or  alkalis  on  either  albumins  or  globulins. 
The  general  properties  of  the  acid-albumin  or  syntonin  and  the  alkali- 
alhumin,  which  are  thereby  respectively  formed  are  as  follows : — 
they  are  insoluble  in  pure  water,  but  are  soluble  in  either  acid  or 
alkali,  and  are  precipitated  by  neutralisation  unless  certain  disturbing 
influences  like  sodium  phosphate  are  present.  They  are  precipitated 
as  globulins  are  by  saturation  with  such  neutral  salts  as  sodium 
chloride  or  magnesium  sulphate.  They  are  not  coagulated  by  heat 
if  in  solution.  In  alkali-albumin  some  of  the  sulphur  in  the  original 
protein  is  removed. 

The  name  albuminate  used  to  be  applied  to  these  substances ;  but  this  is 
an  objectionable  term,  for  these  first  degradation  products  of  protein  hydro- 
lysis are  not  salts,  as  the  termination  -ate  would  imply.  Moreover,  they  are 
obtainable  from  both  albumins  and  globulins.  The  prefix  '  infra- "  (or  pos- 
sibly '  meta-,  which  some  prefer)  may  be  taken  as  an  indication  of  compara- 
tively slight  chemical  alteration. 

A  variety  of  alkali- albumin  (probably  a  compound   containing   a  large 
quantity  of  alkali)    may  be  formed  by  adding  strong  potash  to  undiluted 
white  of  egg.     The  resulting  jelly  is  called  Lieberhiihn'' s  jelly.     A  similar  • 
jelly  is  obtainable  by  adding  strong  acetic  acid  to  undiluted  egg-white. 

The  word  '  albuminate  '  is  also  used  for  compounds  of  protein  with  mineral 
substances.  Thus  if  a  solution  of  copper  sulphate  is  added  to  a  solution  of 
albumin  a  precipitate  of  copper  albuminate  is  formed.  Similarly,  by  the 
addition  of  other  salts  of  the  heavy  metals,  other  metallic  albuminates  are 
obtainable.  The  halogens  (chlorine,  bromine,  iodine)  also  form  albuminates 
in  this  sense,  and  may  be  used  for  the  precipitation  of  proteins. 

It  should  be  noted,  in  conclusion,  that  the  foregoing  classification  of 
proteins  is  mainly  applicable  to  those  of  animal  origin.  The  vegetable  pro- 
teins may  roughly  be  arranged  under  the  same  main  headings,  although  it  is 
doubtful  if  a  real  and  complete  analogy  exists  in  all  cases.  The  cleavage 
products  of  the  vegetable  proteins  are  in  the  main  the  same  as  those  of  the 
animal  proteins,  but  the  quantity  of  each  yielded  is  usually  different. 
Vegetable  proteins,  for  instance,  as  a  rule  give  a  very  much  higher  yield  of 
glutamic  acid  than  do  those  of  animal  origin. 

Further,  there  are  certain  vegetable  proteins  which  have  hitherto  been 
regarded  as  peptones,  but  which  do  not  give  the  biuret  reaction.  It  seems 
impossible  at  present  to  bring  exceptional  substances  of  this  kind  into  any 
general  classification,  and  the  same  is  true  for  those  curious  vegetable  pro- 
teins, such  as  gliadin  from  the  gluten  of  wheat,  and  zein  from  maize,  which 
stand  apart  from  all  other  members  of  the  group  in  being  soluble  in  alcohol. 


LESSON   VI 
FOODS 

A.  Milk.  1.  Examine  a  drop  of  milk  with  the  microscope. 

2.  Note  the  specific  gravity  of  fresh  milk  with  the  lactometer ;  compare 
this  with  the  specific  gravity  of  milk  from  which  the  cream  has  been  removed 
(skimmed  milk).  The  specific  gravity  of  skimmed  milk  is  higher  owing  to 
the  removal  of  the  lightest  constituent — the  cream. 

3.  The  reaction  of  fresh  milk  is  neutral  or  slightly  alkaline  to  litmus. 

4.  Warm  some  milk  in  a  test-tube  to  the  temperature  of  the  body,  and 
add  a  few  drops  of  rennet.  After  standing,  a  curd  is  formed  from  the  con- 
version of  caseinogen,  the  chief  protein  in  milk,  into  casein.  The  casein 
entangles  the  fat  globules.  The  liquid  residue  is  iexmediwhey.  No  curdling 
is  produced  if  the  rennet  solution  is  previously  •  boiled,  because  heat  kilfs 
ferments. 

5.  Take  some  milk  to  which  0*2  per  cent,  of  potassium  oxalate  has  been 
added ;  warm  to  40°  C.  and  add  rennet.  No  curdling  takes  place  because 
the  oxalate  has  precipitated  the  calcium  salts  which  are  necessary  in  the 
coagulation  process. 

Take  a  second  specimen  of  oxalated  milk  and  add  a  few  drops  of  2-per- 
cent, solution  of  calcium  chloride,  and  then  rennet ;  curdling  or  coagulation 
takes  place  if  the  mixture  is  kept  warm  in  the  usual  way. 

6.  To  another  portion  of  warm  milk  diluted  with  water  add  a  few  drops 
of  20-per-cent.  acetic  acid.  A  lumpy  precipitate  of  caseinogen  entanghng 
the  fat  is  formed. 

7.  Filter  off  this  precipitate,  and  in  the  filtrate  test  for  lactose  or  milh 
sugar  by  Trommer's  test  (see  Lesson  II.)  ;  for  lact-albumin  by  boiling,  or  by 
Millon's  reagent  (see  Lesson  IV.) ;  and  for  earthy  (that  is,  calcium  and 
magnesium)  ;pliosphates  by  ammonia,  which  precipitates  these  phosphates. 
Phosphates  may  also  be  detected  by  adding  nitric  acid  and  ammonium 
molybdate  and  boiling  ;  a  yellow  crystalline  precipitate  is  formed. 

8.  Fat  {butter)  may  be  extracted  from  the  precipitate  by  shaking  it  with 
ether ;  on  evaporation  of  the  ethereal  extract  the  fat  is  left  behind,  forming 
a  greasy  stain  on  paper.  The  presence  of  fat  may  also  be  demonstrated  by 
the  black  colour  produced  by  the  addition  of  osmic  acid  to  the  milk. 

9.  Shake  up  a  little  milk  with  twice  its  volume  of  ether  ;  the  opacity  of 
the  milk  remains  nearly  as  great  as  before.  Repeat  this,  but  first  add  to  the 
milk  a  few  drops  of  caustic  potash  before  adding  the  ether  and  then  shake. 
The  milk  which  lies  beneath  the  ethereal  solution  of  fat  becomes  trans- 
lucent. As  a  matter  of  fact  ether  dissolves  the  fat  without  the  addition  of 
alkali,  and  the  opacity  of  milk  is  therefore  not  due  to  the  fat  globules  alone, 
but  largely  to  their  caseinogen  envelope.  The  clearing  which  takes  place 
when  ether  and  alkali  are  added  is  due  to  an  action  of  the  reagents  on  the 
caseinogen. 

10.  Caseinogen,  like  globulins,  is  precipitated  by  saturating  milk  with 
sodium  chloride  or  magnesium  sulphate,  and  by  half  saturation  with 
ammonium  sulphate,  but  differs  from  the  globulins  in  not  being  coagulated 


60  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

by  heat.  The  precipitate  produced  by  saturation  with  salt  floats  to  the 
surface  with  the  entangled  fat,  and  the  clear  salted  whey  is  seen  below  after 
an  hour  or  two. 

B.  Flour. — Mix  some  wheat  flour  with  a  little  water  into  a  stiff  dough. 
Wrap  this  up  in  a  piece  of  muslin  and  knead  it  under  a  tap  or  in  a  capsule  of 
water.  The  starch  grains  come  through  the  holes  in  the  muslin  (identify  by 
iodine  test),  and  an  elastic  sticky  mass  remains  behind.  This  is  a  protein 
called  gluten.  Suspend  a  fragment  of  gluten  in  water ;  add  nitric  acid  and 
boil ;  it  turns  yellow  ;  cool  and  add  ammonia ;  it  turns  orange  (xanthoproteic 
reaction).  Boil  another  fragment  with  Millon's  reagent ;  it  turns  a  brick-red 
colour. 

C.  Bread  contains  the  same  constituents  as  flour,  except  that  some  of  the 
starch  has  been  converted  into  dextrin  and  dextrose  during  baking  (most 
flours,  however,  contain  a  small  quantity  of  sugar).  Extract  breadcruat 
with  cold  water,  and  test  the  extract  for  dextrin  (iodine  test)  and  for 
dextrose  (Trommer's  test).  If  hot  water  is  used,  starch  also  passes  into 
solution. 

D.  Meat. — This  is  our  main  source  of  protein  food.  Cut  up  some  lean 
meat  into  fine  shreds  and  grind  these  up  with  salt  solution.  Filter  and  test 
for  proteins. 

THE    PRINCIPAL    FOOD-STUFFS 

We  can  nov^  proceed  to  apply  the  knowledge  we  have  obtained  of 
the  proteins,  carbohydrates,  and  fats  to  the  investigation  of  some 
important  foods.     The  chief  proximate  principles  in  food  are  : — 

1.  Proteins  \ 

2.  Carbohydrates  h  organic. 

3.  Fats  j 

4.  Water  ). 

5.  Salts  [inorgamc. 

In  milk  and  in  eggs,  which  form  the  exclusive  foods  of  young 
animals,  all  varieties  of  these  proximate  principles  are  present  in 
suitable  proportions.  Hence  they  are  spoken  of  as  perfect  foods. 
Eggs,  though  a  perfect  food  for  the  developing  bird,  contain  too 
little  carbohydrate  for  a  mammal.  In  most  vegetable  foods  carbo- 
hydrates are  in  excess,  while  in  animal  food,  like  meat,  the  proteins. 
Are  predominant.  In  a  suitable  diet  these  should  be  mixed  in 
proper  proportions,  which  must  vary  for  herbivorous  and  carnivorous 
animals.  We  must,  however,  limit  ourselves  to  the  omnivorous 
animal,  man. 

A  healthy  and  suitable  diet  must  possess  the  following  cha- 
racters : — 

1.  It  must  contain  the  proper  amount  and  proportion  of  the 
various  proximate  principles. 

2.  It  must  be  adapted  to  the  climate,  to  the  age  of  the  individual, 
and  to  the  amount  of  work  done  by  him. 


FOODS  51 

3.  The  food  must  contain,  not  only  the  necessary  amount  of  proxi- 
mate principles,  but  these  must  be  present  in  a  digestible  form. 
As  an  instance  of  this,  many  vegetables  (peas,  beans,  lentils)  contain 
even  more  protein  than  beef  and  mutton,  but  are  not  so  nutritious,  as 
they  are  less  digestible,  much  passing  off  in  the  faeces  unused. 

The  nutritive  value  of  a  diet  depends  mainly  on  the  amount  of 
carbon  and  nitrogen  it  contains  in  a  readily  digestible  form.  A  man 
doing  a  moderate  amount  of  work,  and  taking  an  ordinary  diet, 
will  eliminate,  chiefly  from  the  lungs  in  the  form  of  carbonic 
acid,  from  250  to  280  grammes  of  carbon  per  diem.  During  the 
same  time  he  will  eliminate,  chiefly  in  the  form  of  urea  in  the 
urine,  about  15  to  18  grammes  of  nitrogen.  These  substances  are 
derived  from  the  food,  and  from  the  metabolism  of  the  tissues ; 
various  forms  of  energy,  work  and  heat  being  the  chief,  are  simul- 
taneously liberated.  During  muscular  exercise  the  output  of 
carbon  greatly  increases ;  the  increased  excretion  of  nitrogen  is  not 
nearly  so  marked.  Taking,  then,  the  state  of  moderate  exercise,  it  is 
necessary  that  the  waste  of  the  tissues  should  be  replaced  by  fresh 
material  in  the  form  of  food  ;  and  the  proportion  of  carbon  to  nitrogen 
should  be  the  same  as  in  the  excretions :  250  to  15,  or  16*6  to  1. 
The  proportion  of  carbon  to  nitrogen  in  protein  is,  however,  53  to  15, 
or  3*5  to  1.  The  extra  supply  of  carbon  comes  from  non-nitrogenous 
foods — viz.  fat  and  carbohydrate. 

Voit  gives  the  following  daily  diet : — 

Protein  118  grammes. 

Fat  100 

Carbohydrate  333 

Ranke's  diet  closely  resembles  Voit's ;  it  is — 

Protein  100  grammes. 

Fat  100 

Carbohydrate  250        „ 

In  preparing  diet  tables,  such  adequate  diets  as  those  just  given 
should  be  borne  in  mind.  The  following  dietary  (from  G.  N.  Stewart) 
will  be  seen  to  be  rather  more  liberal,  but  may  be  taken  as  fairly 
typical  of  what  is  usually  consumed  by  an  adult  man  in  the  twenty- 
four  hours,  doing  an  ordinary  amount  of  work. 


e2 


52 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


Food-staflE 

Quantity 

Grammes  of 

Nitro- 
gen 

Car- 
bon 

Pro- 
teins 

Fat. 

Carbo- 
hydrates 

Salts 

1 

Metric 

Englisli 

1 

1 

system 

weiglits 

Lean  meat 

250  grammes 

.  9  oz. 

8 

33 

55 

8-5 

0 

4 

Bread       . 

600 

18    „ 

6 

112 

40 

7-5 

245 

6-5 

Milk 

500 

f  pint 

3 

35 

20 

20 

25 

3-5 

Butter      . 

30 

1  oz. 

0 

20 

0 

27 

0 

0-5 

Fat    with 

meat     . 

30 

1   „ 

0 

22 

0 

30 

0 

0 

Potatoes  . 

450 

16    „ 

1-5 

47 

10 

0 

95 

4-5 

Oatmeal  . 

75        „ 

3    „ 

1-7 

30 

10 

4 

48 
413 

2 
21 

20-2 

299 

135 

97 

Eecent  research  has  shown  that,  for  a  certain  time  at  any  rate,  a 
man  will  maintain  his  w^eight  and  health  on  diets  even  scantier  than 
those  of  Voit  and  Ranke.  The  most  convincing  of  these  experiments 
have  been  performed  by  Chittenden  upon  himself  and  others. 
Chittenden  m^ges  that  the  normal  diet  should  contain  only  about 
half  the  customary  quantity  of  protein.  The  body  certainly  does 
not  assimilate  the  larger  amount  usually  taken,  for  the  greater  part 
of  the  nitrogenous  constituents  is  converted  into  amino-acids,  which 
are  rapidly  transformed  by  the  liver  into  urea  and  cast  out  of  the 
body,  leaving  the  non-nitrogenous  remainder  to  be  utilised  as  fats 
and  carbohydrates  are,  for  the  production  of  heat  and  energy. 
"Whether  Chittenden's  views  will  meet  with  general  acceptance  is  at 
present  doubtful,  although  his  work  will  bring  home  to  many  people 
that  temperance  is  necessary  in  food  as  well  as  in  drink.  The 
majority  of  well-to-do  people  cerbainly  eat  an  excess  of  meat,  and  so 
throw  an  unnecessary  strain  upon  their  digestive  and  excretory 
organs.  One  should  hesitate,  however,  in  accepting  Chittenden's 
conclusions  to  the  full,  for  it  is  doubtful  if  the  ^ninimum  is  also  the 
optimum  diet.  It  may  be  that  there  is  a  real  need  for  an  excess  of 
protein  beyond  the  apparent  minimum.  In  diamond  mining  a  large 
quantity  of  earth  must  be  crushed  to  obtain  the  precious  stones.  It 
may  be  that  among  the  many  cleavage  products  of  protein  the 
majority  may  be  compared  to  this  waste  earth,  and  we  get  rid  of 
them  as  quickly  as  possible  in  the  excretions,  but  some  few  may  be 
unusually  precious  for  protein  synthesis  in  the  body,  and  that,  in 
order  to  get  an  adequate  supply  of  these,  a  comparatively  large 
amount  of  protein  must  be  ingested. 


FOODS 


53 


MILK 

Milk  is  often  spoken  of  as  a  *  perfect  food,'  and  it  is  so  for  infants. 
For  those  who  are  older  it  is  so  voluminous  that  unpleasantly  large 
quantities  of  it  would  have  to  be  taken  in  the  course  of  the  day  to 
insure  the  proper  supply  of  nitrogen  and  carbon.  Moreover,  for  adults 
it  is  relatively  too  rich  in  protein  and  fat.  It  also  contains  too  little 
iron  (Bunge)  ;  lience  children  weaned  late  become  anaemic. 

The  microscope  reveals  that  it  consists  of  two  parts  :  a  clear  fluid 
and  a  number  of  minute  particles  that  float  in  it.  These  consist  of 
minute  oil  globules,  varymg  in  diameter  from  00015  to  0*005  milli- 
metre. 

The  milk  secreted  during  the  first  few  days  of  lactation  is  called 
colostrum.     It  contains  very  little  caseinogen,  but  large  quantities 


o 

OoO 
O  " 


AOqO  OAT"    oP^if^   o'O    9^o         V  o^Qn     »  Uo 


0 


"qoco 

00 


^  ^o  o   -O 

Fm.  10. — Microscopic  appearance  of  milk  in  the 
early  stage  of  lactation,  showing  colostrum 
corpuscles  (a)  in  addition  to  fat  globules.  (Yeo.) 


Pig.  11. — a,  b,  colostrum 
corpuscles  with  fine 
and  coarse  fat  globules 
respectively  ;  c,  d,  e, 
pale  cells  devoid  of 
fat.    (Heidenhain.) 


of  globuhn  instead.  Microscopically,  cells  from  the  acini  of  the 
mammary  gland  are  seen,  which  contain  fat  globules  in  their  interior  : 
they  are  called  colostrum  corpuscles. 

Reaction  and  Specific  Gravity. — The  reaction  of  fresh  cow's  milk 
and  of  human  milk  is  amphoteric.  This  is  due  to  the  presence  of 
both  acid  and  alkaline  salts  ;  the  latter  are  usually  in  excess.  Milk 
readily  turns  acid  or  sour  as  the  result  of  fermentative  change,  part 
of  its  lactose  being  transformed  into  lactic  acid  (see  p.  19).  The 
specific  gravity  of  milk  is  usually  ascertained  with  the  hydrometer. 
That  of  normal  cow's  milk  varies  from  1,028  to  1,034.  When  the  milk 
is  skimmed  the  specific  gravity  rises,  owing  to  the  removal  of  the 
Hght  constituent,  the  fat,  to  1,033  to  1,037.     In  all  cases  the  specific 


54  ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 

gravity  of  water,  with  which  other  substances  are  compared,  is  taken 
as  1,000. 

Composition. — Bunge  gives   the  following  table,  contrasting  the 
milk  of  woman  and  cow  ; — 


- 

Woman 

Cow 

Proteins  (chiefly  caseinogen)     . 

Butter  (fat) 

Lactose 

Salts 

Per  cent. 

1-7 
3-4 
6-2 
0-2 

■ 

Per  cent. 

3-5 
3-7 
4-9 
0-7 

Hence,  in  feeding  infants  on  cow's  milk,  it  will  be  necessary  to 
dilute  it,  and  add  sugar  to  make  it  approximately  equal  to  natural 
human  milk. 

The  Proteins  of  Milk. — The  principal  protein  in  milk  is  called 
caseinoge7i :  this  is  the  one  which  is  coagulated  by  rennet  to  form 
casein.  Cheese  consists  of  casein  with  the  entangled  fat.  The  other 
protein  in  milk  is  an  albumin.  It  is  present  in  small  quantities 
only ;  it  differs  in  some  of  its  properties  (specific  rotation,  coagulation, 
temperature,  and  solubilities)  from  serum-albumin ;  it  is  called  lact- 
alhumin. 

The  Coagulation  of  Milk. — Eennet  is  the  agent  usually  employed 
for  this  purpose  :  it  is  a  ferment  secreted  by  the  stomach,  especially 
by  sucking  animals,  and  is  generally  obtained  from  the  calf. 

The  curd  consists  of  the  casein  and  entangled  fat :  the  liquid 
residue  called  whey  contains  the  sugar,  salts,  and  albumin  of  the 
milk.  There  is  also  a  small  quantity  of  a  new  protein  called  whey- 
inotein,  which  differs  from  caseinogen  by  not  being  convertible  into 
casein.  It  is  produced  by  the  decomposition  of  the  caseinogen 
molecule  during  the  process  of  curdling. 

The  curd  formed  in  human  milk  is  more  finely  divided  than  that 
in  cow's  milk :  hence  it  is  more  digestible.  In  feeding  children  and 
invalids  on  cow's  milk,  the  lumpy  condition  of  the  curd  may  be 
obviated  by  the  addition  of  lime  water  or  barley  water  to  the  milk. 

Considerable  discussion  has  taken  place  as  to  whether  the 
caseinogen  of  human  milk  may  not  be  a  different  protein  from  that 
of  cow's  milk,  especially  in  relation  to  the  amount  and  manner  of 
combination  of  its  phosphorus.  The  differences,  however,  appear  to 
be  explicable  on  the  hypothesis  that  they  are  due  to  variations  in  the 
amounts  of  calcium  salts  and  of  citric  acid  which  are  present. 

Caseinogen  itself  may  be  precipitated  by  acids  such  as  acetic  acid, 


FOODS  55 

or  by  saturation  with  neutral  salts  like  sodium  chloride.  This,  how- 
ever, is  not  coagulation,  but  precipitation.  The  precipitate  may  be 
collected  and  dissolved  in  lime  water;  the  addition  of  rennet  then 
produces  coagulation  in  this  solution,  provided  that  a  sufficient 
amount  of  calcium  salts  is  present. 

In  milk  also,  rennet  produces  coagulation,  provided  that  a 
sufficient  amount  of  calcium  salts  is  present.  If  the  calcium  salts 
are  precipitated  by  the  addition  of  potassium  oxalate,  rennet  causes 
no  formation  of  casein.  The  process  of  curdling  in  milk  is  a  double 
one ;  the  first  action  due  to  rennet  is  to  produce  a  change  in  caseino- 
gen  ;  the  second  action  is  that  of  the  calcium  salt,  which  precipitates 
the  altered  caseinogen  as  casein.  In  blood  also  calcium  salts  are 
necessary  for  coagulation  ;  but  there  they  act  in  a  different  w^ay, 
namely,  in  the  production  of  fibrin-ferment  (see  Coagulation  of 
Blood). 

Caseinogen  is  not  coagulable  by  heat.  We  have  already  classed 
it  with  vitellin  as  a  phospho-protein  (see  p.  44). 

Caseinogen,  as  was  originally  pointed  out  by  Hammarsten,  is  a  protein 
with  acid  properties :  it  is  quite  insoluble  in  water,  but  it  forms  soluble 
salts  with  such  metallic  bases  as  potassivim,  sodium,  and  calcium.  The 
caseinogen  as  it  exists  in  ^milk  is  combined  with  calcium  as  calcium  caseino- 
genate.  When  acetic  acid  is  added  to  milk,- we  therefore  get  calcium  acetate, 
and  a  precipitate  of  free  caseinogen.  On  '  dissolving '  this  caseinogen  in  an 
alkali  like  soda  or  potash,  we  have  the  formation  of  sodium  caseinogenate  or 
potassium  caseinogenate,  as  the  case  may  be.  The  precipitate  obtained  in 
milk  by  the  addition  of  alcohol,  or  by  '  salting  out,'  is  not  free  caseinogen, 
but  calcium  caseinogenate.  When  we  add  potassium  oxalate  to  milk,  we 
get  the  reaction  represented  in  the  following  equation : — Calcium  caseino- 
genate +  potassium  oxalate  =  calcium  oxalate  +  potassium  caseinogenate. 
When  we  add  calcium  chloride  to  oxalated  milk,  the  following  equation 
represents  what  occurs  : — Potassium  caseinogenate  +  calcium  chloride  =  cal- 
cium caseinogenate  -^  potassium  chloride. 

Calcium  caseinogenate  forms  an  opalescent  solution  in  water,  and  reacts 
with  the  rennin  ferment.  The  caseinogenates  of  magnesium,  barium,  and 
strontium  have  similar  characters.  The  caseinogenates  of  potassium,  sodium, 
and  ammonium  differ  from  the  above  by  forming  a  nearly  clear  solution  in 
water,  and  they  do  not  react  with  the  rennin  ferment.     (W.  A.  Osborne.) 

The  Fats  of  Milk. — The  chemical  composition  of  the  fat  of  milk 
(butter)  is  very  like  that  of  adipose  tissue.  It  consists  chiefly  of 
palmitin,  stearin,  and  olein.  There  are,  however,  smaller  quantities 
of  fats  derived  from  fatty  acids  lower  in  the  series,  especially  butyrin 
and  caproin.  The  old  statement  that  each  fat  globule  is  surrounded 
by  a  membrane  of  caseinogen  is,  according  to  Eamsden's  recent 
work,  correct.  Milk  also  contains  small  quantities  of  lecithin,  a 
phosphorised  fat ;  of  cholesterin,  an  alcohol  which  resembles  fat  in 
its  solubilities  (see  Bile)  ;  and  a  yellow  fatty  pigment  or  lipochrome. 


56  ESSENTIALS   OF  CHEiMICAL  PHYSIOLOGY 

Milk  Sugar  or  Lactose. — This  is  a  saccharose  (CisHaaOn).  Its 
properties  have  already  been  described  in  Lesson  II.,  p.  19. 

Souring  of  Milk. — When  milk  is  allowed  to  stand,  the  chief  change 
which  it  is  apt  to  undergo  is  a  conversion  of  a  part  of  its  lactose  into 
lactic  acid.  This  is  due  to  the  action  of  micro-organisms,  and  would 
not  occur  if  the  milk  were  contained  in  closed  sterilised  vessels. 
Equations  showing  the  change  produced  are  given  on  p.  19.  When 
souring  occurs,  the  acid  which  is  formed  precipitates  a  portion  of  the 
caseinogen.  This  must  not  be  confounded  with  the  formation  of 
casein  from  caseinogen  which  is  produced  by  rennet.  There  are, 
however,  some  bacterial  growths  which  produce  true  coagulation  like 
rennet. 

Alcoholic  Fermentation  in  Milk.— When  yeast  is  added  to  milk, 
the  sugar  does  not  readily  undergo  the  alcoholic  fermentation.  Other 
somewhat  similar  fungoid  growths  are,  however,  able  to  produce 
the  change,  as  in  the  preparation  of  koumiss ;  the  milk  sugar 
is  first  inverted,  that  is,  dextrose  and  galactose  are  formed  from  it 
(see  p.  19),  and  it  is  from  these  sugars  that  alcohol  and  carbonic  acid 
originate. 

The  Salts  of  Milk. — The  chief  salt  present  is  calcium  phosphate  : 
a  small  quantity  of  magnesium  phosphate  is  also  present.  The  other 
salts  are  chiefly  chlorides  of  sodium  and  potassium. 

EGGS 

In  this  country  the  eggs  of  hens  and  ducks  are  those  particularly 
selected  as  foods.  The  shell  is  made  of  calcareous  matter,  especially 
calcium  carbonate.  The  lohite  is  composed  of  a  richly  albuminous 
fluid  enclosed  in  a  network  of  firmer  and  more  fibrous  material.  The 
amount  of  solids  is  13"3  per  cent.  :  of  this  12-2  is  protein  in  nature. 
The  proteins  are  albumin,  with  smaller  quantities  of  egg-globulin 
and  ovo-mucoid  (p.  45).  The  remainder  is  made  up  of  sugar 
(0*5  per  cent.),  traces  of  fats,  lecithin  and  cholesterin,  and  0*6 
per  cent,  of  inorganic  salts.  The  yolk  is  rich  in  food  materials  for 
the  development  of  the  future  embryo.  In  it  there  are  two  varieties 
of  yolk-spherules,  one  kind  yellow  and  opaque  (due  to  admixture 
with  fat  and  a  yellow  lipochrome),  and  the  other  smaller,  transparent 
and  almost  colourless :  these  are  protein  in  nature,  consisting  of  the 
phospho-protein  called  vitellin  (p.  44).  Small  quantities  of  sugar, 
lecithin,  cholesterin,  and  inorganic  salts  are  also  present. 

The  nutritive  value  of  eggs  is  high,  as  they  are  so  readily  diges- 
tible ;  but  the  more  an  egg  is  cooked  the  more  insoluble  do  its  protein 
constituents  become. 


FOODS 


57 


MEAT 

This  is  composed  of  the  muscular  and  connective  (including 
adipose)  tissues  of  certain  animals.  The  flesh  of  some  animals  is 
not  eaten  ;  in  some  cases  this  is  a  matter  of  fashion  ;  some  flesh,  like 
that  of  the  carnivora,  is  stated  to  have  an  unpleasant  taste ;  and  in 
other  cases  {e.g.  the  horse)  it  is  more  lucrative  to  use  the  animal  as  a 
beast  of  burden. 

Meat  is  the  most  concentrated  and  most  easily  assimilable  of 
nitrogenous  foods.  It  is  our  chief  source  of  nitrogen.  Its  chief  solid 
constituent  is  protein,  and  the  principal  protein  is  myosin.  In  addi- 
tion to  the  extractives  and  salts  contained  in  muscle,  there  is  always 
a  certain  percentage  of  fat,  even  though  all  visible  adipose  tissue  is 
dissected  off.  The  fat-cells  are  placed  between  the  muscular  fibres, 
and  the  amount  of  fat  so  situated  varies  in  different  animals.  It  is 
particularly  abundant  in  pork  ;  hence  the  indigestibility  of  this  form 
of  flesh  ;  the  fat  prevents  the  gastric  juice  from  obtaining  ready 
access  to  the  muscular  fibres. 

The  following  table  gives  the  chief  substances  in  some  of  the 
principal  meats  used  as  food  : — 


Constituents 
Water 

Ox 

Calf 

Pig 

Horse 

Fowl 

Pike 

76-7 

75-6 

72-6 

74-3 

70-8 

79-3 

Sclids 

23-3 

24-4 

27-4 

25-7 

29-2 

20-7 

Proteins,  includ- 

ing gelatin  .     . 

20-0 

19-4 

19-9 

21-6 

22-7 

18-8 

Fat     . 

1-5 

2-9 

6-2 

2-5 

4-1 

0-7 

Carbohydrate      . 

0-6 

0-8 

0-6 

0-6 

1-3 

0-9 

Salts  . 

1-2 

1-3 

1-1 

1-0 

1-1 

0-8 

The  large  percentage  of  water  in  meat  should  be  particularly 
noted  ;  if  a  man  wished  to  take  his  daily  quantity  of  100  grammes 
of  protein  entirely  in  the  form  of  meat,  it  would  be  necessary  for  him 
to  consume  about  500  grammes  {i.e.  a  little  more  than  1  lb.)  of  meat 
per  diem. 

FLOUR 

The  best  wheat  flour  is  made  from  the  interior  of  wheat  grains, 
and  contains  the  greater  proportion  of  the  starch  of  the  grain  and 
most  of  the  protein.  Whole  flour  is  made  from  the  whole  grain 
minus  the  husk,  and  thus  contains,  not  only  the  white  interior,  but 
also  the  harder  and  browner  outer  portion  of  the  grain.  This  outer 
region  contains  a  somewhat  larger  proportion  of  the  proteins  of  the 


58 


ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 


grain.  Whole  flour  contains  1  to  2  per  cent,  more  protein  than  the 
best  white  flour,  but  it  has  the  disadvantage  of  being  less  readily 
digested.  Brown  flour  contains  a  certain  amount  of  bran  in  addi- 
tion ;  it  is  still  less  digestible,  but  is  useful  as  a  mild  laxative,  the 
insoluble  cellulose  mechanically  irritating  the  intestinal  canal  as  it 
passes  along. 

The  best  flour  contains  very  little  sugar.  The  presence  of  sugar 
indicates  that  germination  has  commenced  in  the  grains.  In  the 
manufacture  of  malt  from  barley  this  is  purposely  allowed  to  go  on. 

When  mixed  with  water,  wheat  flour  forms  a  sticky  adhesive 
mass  called  dough.  This  is  due  to  the  formation  of  gluten,  and  the 
forms  of  grain  poor  in  gluten  cannot  be  made  into  dough  (oats,  rice, 
&c.).  Gluten  does  not  exist  in  the  flour  as  such,  but  is  formed  on  the 
addition  of  water  from  the  pre-existing  soluble  proteins  {e.g.  globulins) 
in  the  flour.  It  is  a  mixture  of  several  proteins  (gliadin,  mucedin, 
gluten -fibrin,  &c.). 

The  following  table  contrasts  the  composition  of  some  of  the  more 
important  vegetable  foods  : — 


Constituents 

Wheat 

Barley 

Oats 

Rice 

Lentils 

Peas 

Potatoes 

Water 

13-6 

13-8 

12-4 

13-1 

12-5 

14-8 

76-0 

Protein 

12-4 

11-1 

10-4 

7-9 

24-8 

23-7 

2-0 

Fat    ...         . 

1-4 

2-2 

5-2 

0-9 

1-9 

1-G 

0-2 

Starch 

67-9 

64-9 

57-8 

76-5 

54-8 

49-3 

20-6 

Cellulose    . 

2-5 

5-3 

11-2 

0-6 

3-6 

7-5 

0-7 

Mineral  salts      . 

1-8 

2-7 

3-0 

1-0 

24 

3-1 

1-0 

We  see  from  this  table — 

1.  The  great  quantity  of  starch  always  present. 

2.  The  small  quantity  of  fat  ;  that  bread  is  generally  eaten  with 
butter  is  a  popular  recognition  of  this  fact. 

Protein,  except  in  potatoes,  is  pretty  abundant,  and  especially  so 
in  the  pulses  (lentils,  peas,  &c.).  The  protein  in  the  pulses  is  not 
gluten,  but  consists  of  vitellin  and  globulin-like  substances. 

In  the  mineral  matters  in  vegetables,  salts  of  potassium  and 
magnesium  are,  as  a  rule,  more  abundant  than  those  of  sodium  and 
calcium. 

BREAD 

Bread  is  made  by  cooking  the  dough  of  w^heat  flour  mixed  with 
yeast,  salt,  and  flavouring  materials.  A  ferment  in  the  flour  acts  at 
the  commencement  of  the  process  when  the  temperature  is  kept  a 
little  over  that  of  the  body,  and  forms  dextrin  and  sugar  from  the 


FOODS  59 

starch,  and  then  the  alcoholic  fermentation,  due  to  the  action  of  the 
yeast,  begins.  The  bubbles  of  carbonic  acid,  burrowing  passages 
through  the  bread,  make  it  light  and  spongy.  This  enables  the 
digestive  juices  subsequently  to  soak  into  it  readily  and  affect  all 
parts  of  it.  During  baking  the  gas  and  alcohol  are  expelled  from  the 
bread,  the  yeast  is  killed,  and  a  crust  forms  from  the  drying  of  the 
outer  portions  of  the  dough. 

White  bread  contains,  in  100  parts,  7  to  10  of  protein,  55  of  carbo- 
hydrate, 1  of  fat,  2  of  salts,  and  the  rest  water. 

COOKING  OF   FOOD 

The  cooking  of  foods  is  a  development  of  civilisation,  and  much 
relating  to  this  subject  is  a  matter  of  education  and  taste  rather  than 
of  physiological  necessity.  Cooking,  however,  serves  many  useful 
ends  :-- 

1.  It  destroys  all  parasites  and  danger  of  infection.  This  relates, 
not  only  to  bacterial  growths,  but  also  to  larger  parasites,  such  as 
tapeworms  and  trichinae. 

2.  In  the  case  of  vegetable  foods  it  breaks  up  the  starch  grains, 
bursting  the  cellulose  and  allowing  the  digestive  juices  to  come  into 
contact  with  granulose. 

3.  In  the  case  of  animal  foods  it  converts  the  insoluble  collagen 
of  the  universally  distributed  connective  tissues  into  the  soluble 
gelatin.  The  loosening  of  the  fibres  is  assisted  by  the  formation  of 
steam  between  them.  By  thus  loosening  the  binding  material,  the 
more  important  elements  of  the  food,  such  as  muscular  fibres,  are 
rendered  accessible  to  the  gastric  and  other  juices.  Meat  before  it  is 
cooked  is  generally  kept  a  certain  length  of  time  to  allow  7'igor  mortis 
to  pass  off. 

Of  the  two  chief  methods  of  cooking,  roasting  and  boiling,  the 
former  is  the  more  economical,  as  by  its  means  the  meat  is  first 
surrounded  with  a  coat  of  coagulated  protein  on  its  exterior,  which 
keeps  in  the  juices  to  a  great  extent,  letting  little  else  escape  than 
the  dripping  (fat).  Whereas  in  boiling,  unless  bouillon  and 
bouilli  are  used,  there  is  considerable  waste.  Cooking,  especially 
boiling,  renders  the  proteins  more  insoluble  than  they  are  in  the 
raw  state,  but  this  is  counterbalanced  by  the  other  advantages  that 
cooking  possesses. 

Beef  Tea. — In  making  beef  tea  and  similar  extracts  of  meat  it  is 
necessary  that  the  meat  should  be  placed  in  cold  water,  and  this  is 
gradually  and  carefully  warmed.     In  cooking  a  joint  it  is  usual  to 


60  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

put  the  meat  into  boiling  water  at  once,  so  that  the  outer  part  is 
coagulated,  and  the  loss  of  material  minimised. 

An  extremely  important  point  in  this  connection  is  that  beef  tea 
and  similar  meat  extracts  should  not  be  regarded  as  foods.  They 
are  valuable  as  pleasant  stimulating  drinks  for  invalids,  but  they 
contain  very  little  of  the  nutritive  material  of  the  meat,  their  chief 
constituents,  next  to  water,  being  the  salts  and  extractives  (creatine, 
hypoxanthine,  lactic  acid,  &c.)  of  flesh. 

Many  invalids  restricted  to  a  liquid  diet  get  tired  of  milk,  and 
imagine  that  they  get  sufficient  nutriment  by  taking  beef  tea  instead. 
It  is  very  important  that  this  erroneous  idea  should  be  corrected.  One 
of  the  greatest  difficulties  that  a  physician  has  to  deal  with  in  these 
cases  is  the  distaste  which  many  adults  evince  for  milk.  It  is 
essential  that  this  should  be  obviated  as  far  as  possible  by  preparing 
the  milk  in  different  ways  to  avoid  monotony.  Some  can  take 
koumiss;  but  a  less  expensive  variation  may  be  introduced  in  the 
shape  of  junkets,  which,  although  well  known  in  the  West  of 
England,  are  comparatively  unknown  in  other  parts.  The  preparation 
of  a  junket  consists  of  adding  to  warm  milk  in  a  bowl  or  dish  a  small 
quantity  of  rennet  (Clark's  essence  is  very  good  for  this  purpose)  and 
flavouring  material  according  to  taste.  The  mixture  is  then  put 
aside,  and  in  a  short  time  the  milk  sets  into  a  jelly  (coagulation  of 
casein),  which  may  then  be  served  with  or  without  cream. 

Soup  contains  the  extractives  of  meat,  a  small  proportion  of  the 
proteins,  and  the  principal  part  of  the  gelatin.  The  gelatin  is  usually 
increased  by  adding  bones  and  fibrous  tissue  to  the  stock.  It  is  the 
presence  of  this  substance  which  causes  the  soup  when  cold  to 
gelatinise. 

ACCESSORIES  TO  FOOD 

Among  these  must  be  placed  alcohol,  the  value  of  which  within 
moderate  limits  is  not  as  a  food,  but  as  a  stimulant ;  condiments 
(mustard,  pepper,  ginger,  curry  powder,  &c.),  which  are  stomachic 
stimulants,  the  abuse  of  which  is  followed  by  dyspeptic  troubles ; 
and  tea,  cojfee,  cocoa,  and  similar  drinks.  These  are  stimulants 
chiefly  to  the  nervous  system  ;  tea,  coffee,  mat6  (Paraguay),  guarana 
(Brazil),  cola  nut  (Central  Africa),  bush  tea  (South  Africa),  and  a 
few  other  plants  used  in  various  countries  all  owe  their  chief  pro- 
perty to  an  alkaloid  called  theine  or  caffeine  (C8H,oN402)  ;  cocoa 
to  the  closely  related  alkaloid,  theobromine  (C7H8N4O2) ;  coca  to 
cocaine.  These  alkaloids  are  all  poisonous,  and  used  in  excess,  even 
in  the  form  of  infusions  of  tea  and  coffee,  produce  over-excitment, 


FOODS  61 

loss  of  digestive  power,  and  other  disorders  well  known  to  physicians. 
Coffee  differs  from  tea  in  being  rich  in  aromatic  matters  ;  tea  contains 
a  bitter  principle,  tannin.  To  avoid  the  injurious  solution  of  too 
much  tannin,  tea  should  only  be  allowed  to  infuse  (draw)  for  a  few 
minutes.  Cocoa  is  not  only  a  stimulant,  but  a  food  as  well :  it  con- 
tains about  50  per  cent,  of  fat  and  12  per  cent,  of  protein.  In  cocoa, 
as  manufactured  for  the  market,  the  amount  of  fat  is  reduced  to 
30  per  cent.,  and  the  amount  of  protein  rises  proportionally  to  about 
20  per  cent. 

Green  vegetables  are  taken  as  a  palatable  adjunct  to  other  foods 
rather  than  for  their  nutritive  properties.  Their  potassium  salts  are, 
however,  abundant.  Cabbage,  turnips,  and  asparagus  contain  80  to 
92  water,  1  to  2  protein,  2  to  4  carbohydrates,  and  1  to  1*5  cellulose 
per  cent.  The  small  amount  of  nutriment  in  most  green  foods 
accounts  for  the  large  meals  made  by  and  the  vast  capacity  of  the 
alimentary  canal  of  herbivorous  animals. 


LESSON   VII 
THE  DIGESTIVE  JUICES 

SALIVA 

1.  To  a  little  saliva  in  a  test-tube  add  acetic  acid.  Mucin  is  precipitated 
in  stringy  flakes. 

2.  Filter  some  fresh  saliva  to  separate  ceils  and  mucus,  and  apply  the 
xanthoproteic  or  Millon's  test  to  the  filtrate ;  the  presence  of  protein  is 
shown. 

3.  Put  some  0*5-per-cent.  starch  solution  into  two  test-tubes.  Add  some 
filtered  saliva  to  one  of  them,  and  put  both  in  the  water-bath  at  40°  C. 
After  five  minutes  remove  them  and  test  both  fluids  with  iodine  and 
Trommer's  test.  The  saliva  will  be  found  to  have  converted  the  starch  into 
dextrin  and  sugar  (maltose). 

4.  The  presence  of  potassium  sulphocyanide  (KCNS)  in  saliva  may  be 
shown  by  the  reJl  colour  given  by  a  drop  of  ferric  chloride.  This  colour  is 
discharged  by  mercuric  chloride. 

5.  The  reaction  of  saliva  is  alkaline  to  litmus  paper. 

GASTRIC   DIGESTION 

1.  Half  fill  four  test-tubes— 

A  with  water.  B  with  0-2-per-cent.  hydrochloric  acid.  C  with  0'2- 
per-cent.  hydrochloric  acid.  D  with  solution  of  white  of  egg  (1  to 
10  of  water). 

2.  To  A  add  a  few  drops  of  glycerin  extract  of  stomach  ^  (this  contains 
pepsin)  and  a  piece  of  a  solid  protein  like  fibrin. 

To  B  also  add  pepsin  solution  and  a  piece  of  fibrin. 
To  C  add  only  a  piece  of  fibrin. 

To  D  add  a  few  drops  of  pepsin  solution  and  fill  up  the  tube  with  0-2-per- 
cent, hydrochloric  acid. 

3.  Put  the  tubes  into  the  water-bath  at  40°  C.  and  observe  them  care- 
fully. 

In  A  the  fibrin  remains  unaltered. 

In  B  it  becomes  swollen,  and  gradually  dissolves. 

In  C  it  becomes  swollen,  but  does  not  dissolve. 

4.  After  half  an  hour  examine  the  solution  in  test-tube  B. 

(a)  Colour  some  of  the  liquid  with  litmus  and  neutralise  with  dilute 
alkali.     Acid- albumin  or  syntonin  is  precipitated. 

(b)  Take  another  test-tube,  and  put  into  it  a  drop  of  1-per-cent.  solution  of 
copper  sulphate  ;  empty  it  out  so  that  the  merest  trace  of  copper  sulphate 
remains  adherent  to  the  wall  of  the  tube ;  then  add  the  solution  from  test- 
tube   B  and  a  few  drops  of  strong  caustic  potash.    A  pink  colour  (biuret 

'  Benger's  liquor  pepticus  may  be  used  instead  of  the  glycerin  extract  of 
stomach. 


THE   DIGESTIVE  JUICES  63 

reaction)  is  produced.     This  should  be  carefully  compared  with  the  violet 
tint  given  by  unaltered  albumin. 

(c)  To  a  third  portion  of  the  fluid  in  test-tube  B  add  a  drop  of  nitric  acid 
proteoses   or   propeptones    are   precipitated.     This   precipitate   dissolves  on 
heating  and  reappears  on  cooling. 

5.  Repeat  these  three  tests  with  the  digested  white  of  egg  in  test-tube  D. 

6.  Examine  an  artificial  gastric  digestion  which  has  been  kept  a  week. 
Note  the  absence  of  putrefactive  odour ;  in  this  it  contrasts  very  forcibly 
with  an  artificial  pancreatic  digestion  under  similar  circumstances. 


FERMENTS 

The  word  fermentation  was  first  applied  to  the  change  of  sugar 
into  alcohol  and  carbonic  acid  by  means  of  yeast.  The  evolution  of 
carbonic  acid  causes  frothing  and  bubbling ; 
hence  the  term  *  fermentation.'  The  agent  yeast 
which  produces  this  is  called  the  ferment. 
Microscopic  investigation  shows  that  yeast  is 
composed  of  minute  rapidly  growing  unicellular 
organisms  (torulse)  belonging  to  the  fungus 
group  of  plants. 

The   souring   of   milk,  the  transformation  of     ,,    ,„    ^„    ,,, 

"  '  Pig.  12.— Cells  of  the  yeast 

urea  into  ammonium  carbonate  in  decomposing        plant  in  process  of  bud- 

.  .  -IS  fluig>    between    which 

urine,  and  the  formation  of  vinegar  (acetic  acid)  are  some  bacteria. 
from  alcohol  are  produced  by  the  growth  of  very 
similar  organisms.  The  complex  series  of  changes  known  as  putre- 
faction, which  are  accompanied  by  the  formation  of  malodorous 
gases,  and  which  are  produced  by  the  growth  of  various  forms  of 
bacteria,  also  come  into  the  same  category. 

That  the  change  or  fermentation  is  produced  by  these  organisms 
is  shown  by  the  fact- that  it  occurs  only  when  the  organisms  are 
present,  and  stops  when  they  are  removed  or  killed  by  a  high  tem- 
perature or  by  certain  substances  (carbolic  acid,  mercuric  chloride, 
&c.)  called  antiseptics.  The  organisms  produce  fermentative  effects 
by  shedding  out  soluble  ferments  or  enzymes. 

The  *  germ  theory  '  of  disease  explains  the  infectious  diseases  by 
considering  that  the  change  in  the  system  is  of  the  nature  of  fermen- 
tation, and,  like  the  others  we  have  mentioned,  produced  by  microbes  ; 
the  transference  of  the  bacteria  or  their  spores  from  one  person  to 
another  constitutes  infection.  The  poisons  produced  by  the  growing 
bacteria  appear  to  be  either  alkaloidal  (ptomaines)  or  protein  in 
nature.  The  existence  of  poisonous  proteins  is  a  very  remarkable 
thing,  as  no  profound  chemical  differences  have  yet  been  shown  to 
exist  between  them  and  those  which  are  not  poisonous,  but  which  are 


64 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


useful  as  foods.     Snake  venom  is  an  instance  of  a  very   virulent 
poison  of  protein  nature. 

There  is  another  class  of  chemical  transformations  which  at  first 
sight  differ  very  considerably  from  all  of  these.  They,  however, 
resemble  these  fermentations  in  the  fact  that  they  occur  indepen- 
dently of  any  apparent  change  in  the  agents  that  produce  them. 
The  agents  that  produce  them  are  not  living  organisms,  but  chemical 


Fig.  13.— Typical  forms  of  Scbizomycetes  (after  Zopf )  :  a,  micrococcus; 
terium ;  d,  bacillus ;  e,  Clostridium ;  /,  monas  Okeuii ;  ff,  leptotlirix ;  h, 
I,  spiruliua  ;  m,  spiromonas  ;  n,  spirochsete ;  o,  cladothrix. 


macrococcus  ;  c,  bac- 
vibrio  ;  /fc,  spirillum  ; 


substances,  the  result  of  the  activity  of  living  cells.     The  change  of 
starch  into  sugar  by  the  ptyalin  of  the  saliva  is  an  instance. 
Ferments  may  therefore  be  divided  into  two  classes : — 

1.  The  organised  ferments — torulaB,  bacteria,  &c. 

2.  The  unorganised  ferments  or  enzymes — like  ptyalin. 

Each  may  be  again  subdivided  according  to   the  nature  of  the 
chemical  change  produced. 

In  digestion,  the  study  of  which  we  are  just  commencing,  it  is  the 


THE  DIGESTIVE  JUICES 


65 


unorganised  ferments  with  the  action  of  which  we  have  chiefly  to 
deal.     The  unorganised  ferments  may  be  classified  as  follows  : — 

(a)  Amylolytic — those  which  change  amyloses  (starch,  glycogen) 
into  sugars.     Examples  :  ptyalin,  diastase,  amylopsin. 

(b)  Proteolytic — those  which  change  native  proteins  into  proteoses 
and  peptones.     Examples  :  pepsin,  trypsin. 

(c)  Steatolytic  or  lipolytic — those  which  split  fats  into  fatty  acids 
and  glycerin.     An  example,  steapsin,  is  found  in  pancreatic  juice. 

(d)  Inversive — those  which  convert  saccharoses  (cane  sugar, 
maltose,  lactose)  into  glucose.  Examples :  invertin  of  intestinal 
juice  and  of  yeast  cells. 

A 


Fig.  14. — Bacillus  anthracis,  the  agent  that  produces  anthrax  or  splenic  fever  (iloch)  :  A,  bacilli 
mingled  with  blood  corpuscles  from  the  blood  of  guinea-pig,  some  of  the  bacilli  dividing  ;  B,  the 
same  after  three  hours'  culture  in  a  drop  of  aqueous  humour.  They  grow  out  into  long  leptothrix- 
]ike  filaments,  which  subsequently  divide  up,  and  spores  are  developed  in  the  segments. 

(e)  Coagulative — those  which  convert  soluble  into  insoluble 
proteins.     Examples  :  rennet,  fibrin  ferment. 

Most  ferment  actions  are  hydrolytic — i.e.  water  is  added  to  the 
material  acted  on,  which  then  splits  into  new  materials.  This  is  seen 
by  the  following  examples  : — 

1.  Conversion  of  cellulose  into  carbonic  acid  and  marsh  gas 
(methane)  by  putrefactive  organisms. 

(C6Hio05)7i+?iH20=3nC02  +  3wCH4 


[cellulose] 


[water] 


[carbonic 
acid] 


[methane] 


2.  Inversion  of  cane  sugar  by  the  unorganised  ferment  invertin : — 

Ci2H220]i,+H20  =  G6H,206  +  C6Hi206 

[cane  sugar]        [water]      [dextrose]  [levulose] 


66  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

It  appears  also  that  some  enzymes  are  oxygen  carriers  and  pro- 
duce oxidation  :  they  are  termed  oxidases. 

A  remarkable  fact  concerning  the  ferments  is,  that  the  substances 
they  produce,  in  time  put  a  stop  to  their  activity ;  thus  in  the  case 
of  the  organised  ferments  the  alcohol  produced  by  yeast,  the  phenol, 
cresol,  &c.,  produced  by  putrefactive  organisms  from  proteins,  first 
stop  the  growth  of,  and  ultimately  kill,  the  organisms  which  produce 
them.  In  the  case  of  the  unorganised  ferments  the  products  of  their 
activity  hinder  and  finally  stop  their  action,  but  on  the  removal  of 
these  products  the  ferments  resume  work. 

This  fact  suggested  to  Croft  Hill  the  question  whether  ferments 
will  act  in  the  reverse  manner  to  their  usual  action ;  and  in  the 
case  of  one  ferment,  at  any  rate,  he  found  this  to  be  the  case. 
Inverting  ferments,  as  we  have  just  seen,  usually  convert  a 
disaccharide  into  monosaccharides.  One  of  these  inverting  ferments, 
called  maltase,  converts  maltose  into  dextrose.  If,  however,  the 
ferment  is  allowed  to  act  on  strong  solutions  of  dextrose,  it  converts 
a  small  proportion  of  that  sugar  back  into  maltose.  '  Eeversible 
action'  has  since  this  been  shown  to  occur  in  the  case  of  other 
enzymes. 

Ferments  act  best  at  a  temperature  of  about  40°  C.  Their 
activity  is  stopped,  but  the  ferments  are  not  destroyed,  by  cold  ;  it  is 
stopped  and  the  ferments  killed  by  great  heat.  A  certain  amount 
of  moisture  and  oxygen  is  also  necessary ;  there  are,  however,  certain 
micro-organisms  that  act  without  free  oxygen  :  these  are  called 
anaerobic,  in  contradistinction  to  those  that  require  oxygen,  and 
which  are  therefore  called  aerobic. 

The  chemical  nature  of  the  enzymes  is  very  difficult  to  investigate ; 
they  are  substances  that  to  a  great  extent  elude  the  grasp  of  the 
chemist.  So  far  research  has  taught  us  that  they  are  either  protein 
in  nature  or  are  substances  closely  allied  to  the  proteins. 

The  distinction  between  organised  ferments  and  enzymes  is,  how- 
ever, more  apparent  than  real ;  for  the  micro-organisms  exert  their 
action  by  enzymes  that  they  secrete.  This  has  long  been  known 
in  connection  with  the  invertin  of  yeast  and  with  the  enzyme 
secreted  by  the  micrococcus  ureae  which  converts  urea  into  ammonium 
carbonate.  In  recent  years  Buchner  by  crushing  yeast  cells  succeeded 
in  obtaining  from  them  an  enzyme  which  produces  the  alcoholic 
fermentation,  and  there  is  now  no  doubt  that  what  is  true  for  yeast 
is  true  for  all  the  organised  ferments ;  in  several  cases  this  has 
already  been  proved  experimentally. 

The  view  at  present  current  regarding  ferment  action  is  that  they 


THE  DIGESTIVE  JUICES  67 

are  catalysing  agents.  That  is  to  say,  their  presence  induces  a 
chemical  reaction  to  occur  rapidly  which  in  their  absence  also  occurs, 
but  so  slowly  that  any  action  at  all  is  difficult  to  discover.  To  use 
the  technical  phrase,  their  action  is  to  increase  the  velocity  of  chemical 
reactions.  The  enzymes  are  catalysts  of  a  colloidal  nature,  and 
certain  properties,  in  which  they  differ  from  most  inorganic  catalysts 
are  due  to  this  circumstance. 

THE  SALIVA 

The  secretion  of  saliva  is  a  reflex  action ;  the  taste  or  smell  of 
food  excites  the  nerve-endings  of  the  afferent  nerves  (glossopharyngeal 
and  olfactory)  ;  the  efferent  or  secretory  nerves  are  contained  in  the 
chorda  tympani  (a  branch  of  the  seventh  cranial  nerve)  which 
supplies  the  submaxillary  and  sublingual,  and  in  a  branch  of  the 
glossopharyngeal  which  suppHes  the  parotid.  The  sympathetic 
branches  which  supply  the  blood-vessels  with  constrictor  nerves 
contain  in  some  animals  secretory  fibres  also. 

The  parotid  gland  is  called  a  serous  or  albuminous  gland  ;  before 
secretion  the  cells  of  the  acini  are  swollen  out  with  granules ;  after 
secretion  has  occurred  the  cells  shrink,  owing  to  the  granules  having 
been  shed  out  to  contribute  to  the  secretion  (see  fig.  15). 

The  submaxillary  and  sublingual  glands  are  called  mucous 
glands:  their  secretion  contains  mucin.  Mucin  is  absent  from 
parotid  saliva.  The  granules  in  the  cells  are  larger  than  those  of 
the  parotid  gland  :  they  are  composed  of  mucinogen,  the  precursor 
of  mucin  (see  fig.  16). 

In  a  section  of  a  mucous  gland  prepared  in  the  ordinary  way  the 
mucinogen  granules  are  swollen  out,  and  give  a  highly  refracting 
appearance  to  the  mucous  acini  (see  fig.  17). 

COMPOSITION   OF   SALIVA 

On  microscopic  examination  of  mixed  saliva  a  few  epithelial 
scales  from  the  mouth  and  salivary  corpuscles  from  the  tonsils 
are  seen.  The  liquid  is  transparent,  slightly  opalescent,  of  slimy 
consistency,  and  may  contain  lumps  of  nearly  pure  mucin.  On 
standing  it  becomes  cloudy  owing  to  the  precipitation  of  calcium 
carbonate,  the  carbonic  acid  which  held  it  in  solution  as  bicarbonate 
escaping. 

Of  the  three  forms  of  saliva  which  contribute  to  the  mixture 
found  in  the  mouth,  the  sublingual  is  richest  in  soHds  (2*75  per  cent.). 

f2 


68  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

The  submaxillary  saliva  comes  next  (2*1  to  2*5  per  cent.).  When 
artificially  obtained  by  stimulation  of  nerves  in  the  dog  the  saliva 
obtained  by  stimulation  of  the  sympathetic  is  richer  in  sohds  than 
that  obtained  by  stimulation  of  the  chorda  tympani.  The  parotid 
saliva  is  poorest  in  total  solids  (0*3  to  0*5  per  cent.),  and  contains  no 
mucin.  Mixed  saliva  contains  in  man  an  average  of  about  0*5  per 
cent,  of  solids  :  it  is  alkaline  in  reaction,  due  to  the  salts  in  it ;  and 
has  a  specific  gravity  of  1002  to  1006. 

The    solid   constituents   dissolved   in    saliva    may   be   classified 
thus : — 

a.  Mucin  :  this  may  be  precipitated  by  acetic  acid. 

h.  Ptyalin  :  an  amylolytic  ferment. 

c.  Protein  :  of  the  nature  of  a  globulin. 

d.  Potassium  sulphocyanide. 

e.  Sodium  chloride  :  the  most  abundant  salt. 
/.  Other  salts  :  sodium  carbonate ;  calcium  phosphate 

and   carbonate ;    magnesium  phosphate ;    potassium 
chloride. 


Organic 


Inorganic 


THE  ACTION   OF   SALIVA 

The  action  of  saliva  is  twofold,  physical  and  chemical. 

The  physical  use  of  saliva  consists  in  moistening  the  mucous 
membrane  of  the  mouth,  assisting  the  solution  of  soluble  substances 
in  the  food,  and  in  virtue  of  its  mucin  lubricating  the  bolus  of  food 
to  facilitate  swallowing. 

The  chemical  action  of  saliva  is  due  to  its  active  principle, 
ptyalin.  This  substance  belongs  to  the  class  of  unorganised  ferments 
or  e7izymes,  and  to  that  special  class  of  unorganised  ferments  which 
are  called  amylolytic  (starch-splitting)  or  diastatic  (resembling 
diastase,  the  similar  ferment  in  germinating  barley  and  other  grains). 

The  starch  is  first  split  into  dextrin  and  maltose ;  the  dextrin  is 
subsequently  converted  into  maltose  also  :  this  occurs  more  quickly 
with  erythro-dextrin,  which  gives  a  red  colour  with  iodine,  than  with 
the  other  variety  of  dextrin  called  achroo-dextrin,  which  gives  no 
colour  with  iodine.  Brown  and  Morris  give  the  following  equa- 
tion > 

10(C6H,o05).  +  ^nB.,0 

[starch]  .    [water] 

=47iCi2H220n  +  {Ce^ioO,)n  +  (CeH.oOs),, 

[maltose]  [achroo-dextriil]        [erythro-dextrin] 

Ptyalin  acts  in  a  similar  way,  but  more  slowly  on  glycogen :  it 


THE   DIGESTIVE   JUICES 


69 


has  no  action   on  cellulose ;    hence  it   is   inoperative   on   uncooked 
starch  grains,  for  in  these  the  cellulose  layers  are  intact. 


Fig.  15. — Alveoli  of  serous  gland ;    A,  loaded  befoi'e  secretion ;  B,  after  a  short  period  of  active 
secretion  ;  C,  after  a  prolonged  period.    (Laugley.) 


Fig.  16.— Mucous  cells  from  a  fresli  submaxillary  gland  of  dog  :  a,  loaded  with  muciuogen  granules 
before  secretion  ;  6,  after  secretion  :  the  granules  are  fewer,  especially  at  the  attached  border  of 
the  cell ;  a'  and  5'  represent  cells  in  a  loaded  and  discharged  condition  respectively  which  have 
been  irrigated  with  water  or  dilute  acid.  The  mucous  granules  are  swollen  into  a  transparent 
mass  of  mucin  traversed  by  a  network  of  protoplasmic  cell-substance.    (Foster,  after  Langley.) 


Fig.  17. — Section  of  part  of  the  human  submaxillary  gland.    (Heidenhain.)    To  the  right  is  a  group 
of  mucous  alveoli,  to  the  left  a  group  of  serous  alveoli. 

Ptyalin  acts  best  about   the  temperature  of   the  body  (35-40°), 
and   in   a   neutral   medium ;   a  small    amount  of   alkali  makes  but 


70  ESSENTIALS  OF  CHEMICAL  PHYSIOLOaY 

little  difference ;  a  very  small  amount  of  acid  stops  its  activity. 
The  conversion  of  starch  into  sugar  by  saliva  in  the  stomach 
continues  for  a  considerable  time,  for  the  swallowed  masses  which 
fall  into  the  fundus  of  the  stomach  are  not  subjected  to  peristalsis 
and  admixture  with  gastric  juice  until  a  later  stage  in  digestion ; 
the  hydrochloric  acid  which  is  poured  out  by  the  gastric  glands  j5rst 
neutralises  the  saliva  and  combines  with  the  proteins  in  the  food  ;  but 
immediately  free  hydrochloric  acid  appears  the  ptyalin  is  destroyed, 
so  that  it  does  not  resume  work  even  when  the  semi-digested  food 
once  more  becomes  alkaline  in  the  duodenum. 


f  THE   SECRETION   OF   GASTRIC  JUICE 

The  nice  secreted  by  the  glands  in  the  mucous  membrane  of  the 
stomach  varies  in  composition  in  the  different  regions,  but  the  mixed 
juice  is  a  solution  of  a  proteolytic  ferment  called  pepsin  in  a  saline 
solution,  which  also  contains  a  little  free  hydrochloric  acid. 

The  gastric  juice  can  be  obtained  during  the  life  of  an  animal  by 
means  of  a  gastric  fistula.  Gastric  fistulas  have  also  been  made  in 
human  beings,  either  by  accidental  injury  or  by  surgical  operations. 
The  most  celebrated  case  is  that  of  Alexis  St.  Martin,  a  young 
Canadian  who  received  a  musket  v^ound  in  the  abdomen  in  1822. 
Observations  made  on  him  by  Dr.  Beaumont  formed  the  startmg- 
point  for  our  correct  knowledge  of  the  physiology  of  the  stomach 
and  its  secretion. 

We  now  make  artificial  gastric  juice  by  mixing  weak  hydrochloric 
acid  (0*2  to  0*4  per  cent.)  with  a  glycerin  or  aqueous  extract  of  the 
stomach  of  a  recently  killed  animal.     This  acts  like  the  normal  juice. 

Three  kinds  of  glands  are  distinguished  in  the  stomach,  w^hich 
differ  from  each  other  in  their  position,  in  the  character  of  their 
epithelium,  and  in  their  secretion.  The  cardiac  glands  are  simple 
tubular  glands  quite  close  to  the  cardiac  orifice.  The  fundus  glands 
are  those  situated  in  the  remainder  of  the  cardiac  half  of  the 
stomach  :  their  ducts  are  short,  their  tubules  long  in  proportion. 
The  latter  are  filled  with  polyhedral  cells,  only  a  small  lumen  being 
left :  they  are  more  closely  granular  than  the  corresponding  cells  in 
the  pyloric  glands.  They  are  called  i)rinciijal  or  central  cells. 
Between  them  and  the  basement  membrane  of  the  tubule  are  other 
cells  which  stain  readily  with  aniline  dyes.  They  are  called  imrietal 
or  oxyntic  {i.e.  acid-forming)  cells.  The  pyloric  glands,  in  the  pyloric 
half  of  the  stomach,  have  long  ducts  and  short  tubules  lined  with 
cubical  cells.     There  are  no  parietal  cells. 


THE   DIGESTIVE  JUICES 


71 


Fig.  19. — A  pyloric  glaud  from  a 
section  of  the  dog's  stomach 
(Ebstein)  :  m,  month ;  n,  neck ; 
tr,  a  deep  portion  of  tubule  cut 
transversely. 


Fig.  18.— a  fundus  glund  from  the  dog's  stomach  (Klein)  :  d,  duct  or  mouth  of  the  gland  ;  b,  base 
of  one  of  its  tubules ;  on  tlie  right  the  base  of  a  tubule  is  more  highly  magnified ;  c,  central 
cell ;  p,  parietal  cell. 


72 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOaY 


The  central  cells  of  the  fundus  glands  and  the  cells  of  the  pyloric 
glands  are  loaded  with  granules.  During  secretion  they  discharge 
their  granules,  those  that  remain  being  chiefly  situated  near  the 
lumen,  leaving  in  each  cell  a  clear  outer  zone  (see  fig.  20).  These 
are  the  cells  that  secrete  the  pepsin.  Like  secreting  cells  generally, 
they  select  certain  materials  from  the  lymph  that  bathes  them  : 
these  materials  are  worked  up  by  the  protoplasmic  activity  of  the 

cells  into  the  secretion,  which  is  then  dis- 
charged into  the  lumen  of  the  gland.  The 
most  important  substance  in  a  digestive 
secretion  is  the  ferment.  In  the  case  of  a 
gastric  juice  this  is  pepsin.  We  can  trace 
an  intermediate  step  in  this  process  by  the 
presence  of  the  granules.  The  granules 
are  not,  however,  composed  of  pepsin,  but 
of  a  mother-substance,  which  is  readily 
converted  into  pepsin.  We  shall  find  a 
similar  ferment  precursor  in  the  cells  of 
the  pancreas,  and  the  term  zymogen  is 
applied  to  these  ferment  precursors.  The 
zymogen  in  the  gastric  cells  is  called  pep- 
sinogen.  The  rennet-ferment  or  rennin  that 
causes  the  curdling  of  milk  is  distinct  from 
pepsin,*  and  is  preceded  by  another  zymo- 
gen ;  it  is,  however,  formed  by  the  same 
cells. 

The  parietal  cells  are  also  called  oxyntic 
cells,  because  they  secrete  the  hydrochloric 
acid  of  the  juice.  Heidenhain  succeeded 
in  making  in  one  dog  a  cul-de-sac  of  the 
fundus,  in  another  of  the  pyloric  region  of 
the  stomach;  the  former  secreted  a  juice 
containing  both  acid  and  pepsin ;  the  latter, 
parietal  cells  being  absent,  secreted  a  viscid 
alkaline  juice  containing  pepsin.  The  for- 
mation of  a  free  acid  from  the  alkaline 
blood  and  lymph  is  an  important  but  puzzling  problem.  There 
is  no  doubt  that  it  is  formed  from  the  chlorides  of.  the  blood 
and  lymph,  and  of  the  chemical  theories  advanced  as  to  how 
this  is  done,  Maly's  is  the  most  satisfactory.     He  considers  that  the 

'  The  individuality  of  rennin  has  been  questioned  by  Pawlow,  who  regards  its 
action  as  a  phase  of  pepsin  activity. 


Fig.  20.— a  fundus  gland  of  simple 
form  from  the  bat's  stomach. 
Osmic  acid  preparation  (Lang- 
ley)  :  c,  columnar  epithelium 
of  the  surface ;  n,  neck  of  the 
gland,  with  central  and  parietal 
cells  ;  /,  base  occupied  only 
by  principal  or  central  cells, 
which  exhibit  the  granules 
accumulated  towards  the  lumen 
of  the  gland. 


THE   DIGESTIVE   JUICES 


73 


acid  originates  by  the  interaction  of   sodium   chloride  and  sodium 
dihydrogen  phosphate,  as  is  shown  in  the  following  equation  :  — 

NaH2P04  +  NaCl=Na2HP04  +  HCl 


[sodium  di- 

[sodium 

[disodium 

[hydro- 

hydrogeu 

chloride] 

hydrogen 

chloric 

phosphate] 

phosphate] 

acid] 

The  sodium  dihydrogen  phosphate  in  the  above  equation  is  prob- 
ably derived  from  the  interaction  of  the  disodium  hydrogen  phos- 
phate and  the  carbonic  acid  of  the  blood,  thus  : — 

NasHPO^  +  CO2  +  H20=NaHC03  +  NaH2P04. 

Other  theories  have  tried  to  explain  the  formation  of  such  a  strong 
acid  as  hydrochloric  by  the  law  of  *  mass  action.'  We  know  that  by 
the  action  of  large  quantities  of  carbonic  acid  on  salts  of  the  mineral 
acids  the  latter  may  be  liberated  in  small  quantities.  We  know, 
further,  that  small  quantities  of  acid  ions  may  be  continually  formed 
in  the  organism  by  ionisation.  But  in  every  case  we  can  only  make 
use  of  these  explanations  if  we  assume  that  the  small  quantities  of 
acid  are  carried  away  as  soon  as  they  are  formed,  and  thus  give  room 
for  the  formation  of  fresh  acid.  Even  then  it  is  impossible  to 
explain  the  whole  process.  A  specific  action  of  the  cells  is  no  doubt 
exerted,  for  these  reactions  can  hardly  be  considered  to  occur  in  the 
blood  generally,  but  rather  in  the  oxyntic  cells,  which  possess  the 
necessary  selective  powers  in  reference  to  the  constituents  of  the 
blood,  and  the  hydrochloric  acid,  as  soon  as  it  is  formed,  passes 
into  the  secretion  of  the  gland  in  consequence  of  its  high  power  of 
diffusion. 


COMPOSITION   OF  GASTRIC  JUICE 

The   following   table   gives    the   percentage   composition   of  the 
gastric  juice  of  man  and  dog  : — 


Constituents 

Human 

Dog 

Water          .... 

99-44 

97-30 

Organic  sul 
^pepsin) 

stanc 

es  (chiefly 

0-32 
0-20 

1-71 
0-50 

CaCL.  . 

0-006 

0-06 

NaCl  . 

0-14 

0-25 

KCl     . 

0-05 

0-11 

NH.Cl 
Ca3(P0,), 
Mg3(P0,), 
FePO, 

0-01 

0-05 
0-17 
0-02 
0-008 

1 
1 

74  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

One  sees  from  this  how  much  richer  in  all  constituents  the  gastric 
juice  of  the  dog  is  than  that  of  man.  Carnivorous  animals  have 
always  a  more  powerful  gastric  juice  than  other  animals  :  they  have 
more  work  for  it  to  do  ;  but  the  great  contrast  seen  in  the  table  is, 
no  doubt,  partly  due  to  the  fact  that  the  persons  from  whom  it  has 
been  possible  to  collect  gastric  juice  have  been  invalids.  In  the 
foregoing  table  one  also  sees  the  great  preponderance-  of  chlorides 
over  other  salts  :  apportioning  the  total  chlorine  to  the  various 
metals  present,  that  which  remains  over  must  be  combined  with 
hydrogen  to  form  the  free  hydrochloric  acid  of  the  juice. 

Pepsin  stands  apart  from  nearly  all  other  ferments  by  requiring 
an  acid  medium  in  order  that  it  may  act.  A  compound  of  the  two 
substances  called  pepsin-hydrochloric  acid  is  the  really  active  agent. 
Other  acids  may  take  the  place  of  hydrochloric  acid,  but  none  act  so 
well.  Lactic  acid  is  often  found  in  gastric  juice ;  this,  however,  is 
derived  by  fermentative  processes  from  the  food. 

Pawlow  has  shown  that  in  dogs  the  secretory  fibres  for  the  gastric 
glands  are  contained  in  the  vagus  nerves. 

By  an  ingenious  surgical  operation  he  succeeded  in  separating  off 
from  the  stomach  a  diverticulum  which  pours  its  secretion  through  an 
opening  in  the  abdominal  wall.  This  small  stomach  was  found  to 
act  in  every  way  like  the  main  stomach  of  the  animal.  The  pure  juice 
so  obtained  is  clear  and  colourless :  it  has  a  specific  gravity  of  1003 
to  1006.  It  is  feebly  dextro -rotary,  and  gives  some  of  the  protein 
reactions.  It  contains  from  0*4  to  0'6  per  cent,  of  hydrochloric  acid. 
It  is  strongly  proteolytic,  and  inverts  cane  sugar.  When  cooled  to 
0°  C.  it  deposits  a  fine  precipitate  of  pepsin  :  this  settles  in  layers, 
and  the  layers  first  deposited  contain  most  of  the  acid,  which  is 
loosely  combined  with  and  carried  down  by  the  pepsin.  Pepsin  is 
also  precipitable  by  saturation  with  ammonium  sulphate  (Kiihne). 
Elementary  analysis  gave  the  following  results  : — 

Pepsin  precipitated  by  cold —  Precipitated  by  (NHJ2SO4 

Carbon    .  .         .     50-73  per  cent,  j  50*37 

Hydrogen  .         .       7'23       „  !  6-88 

Chlorine.  .  I'Ol  to  1-17       „  '  0*89 

Sulphur  .  .         .       0-98       „  1-34 

Nitrogen .         .         .  not  estimated  14*55  to  15*0 

Oxygen    .         .         .  the  remainder  I  the  remainder. 

The  juice  is  most  abundant  in  the  early  periods  of  digestion,  but 
it  continues  to  be  secreted  in  declining  quantity  as  long  as  any  food 
remains  to  be  dealt  with.  When  there  is  no  food  given  there  is  no 
juice.     But  sham  feeding  with  meat  will  cause  it  to  flow. 

The  larger  the  proportion  of  protein  in  the  diet,  the  more  abundant 


THE  DiaESTIVE  JUICES  75 

and  active  is  the  juice  secreted,  provided  the  animal  is  hungry :  the 
psychical  element  is  of  great  importance. 

THE  ACTION   OF   GASTRIC  JUICE 

The  principal  actions  of  the  gastric  juice  have  been  already  prac- 
tically studied  :  the  action  of  pepsin  in  converting  the  proteins  of  the 
food  into  the  diffusible  peptones  is  its  chief  action.  The  curdling  of 
milk  by  rennet  will  be  found  described  in  Lesson  VI. 

There  is  still  further  action — that  is,  the  gastric  juice  is  anti- 
septic ;  putrefactive  processes  do  not  normally  occur  in  the  stomach, 
and  the  organisms  that  produce  such  processes,  many  of  which  are 
swallowed  with  the  food,  are  in  great  measure  destroyed,  and  thus 
the  body  is  protected  from  them.  The  acid  is  the  agent  in  the  juice 
that  possesses  this  power. 

The  formation  of  peptones  is  a  process  of  hydrolysis ;  peptones 
may  be  formed  by  other  hydrating  agencies  like  superheated  steam 
and  heating  with  dilute  mineral  acids.  There  are  certain  inter- 
mediate steps  in  this  process  ;  the  intermediate  substances  are  called 
propeptones  or  proteoses.  The  word  *  proteose  '  is  the  best  to  employ : 
it  includes  the  albumoses  (from  albumin),  globuloses  (from  globulin), 
vitelloses  (from  vitellin),  &c.  Similar  substances  are  also  formed 
from  gelatin  (gelatoses)  and  elastin  (elastoises). 

Another  intermediate  step  in  gastric  digestion  is  .acid-albumin  or 
syntonin.  In  classifying  the  products  of  digestion  it  will  be  con- 
venient to  take  albumin  as  our  example,  but  we  must  remember  that 
globulin,  myosin,  and  all  the  other  proteins  form  corresponding 
products.  The  products  of  digestion  may  be  classified  according  to 
the  order  in  which  they  are  formed  as  follows  : — 

1.  Acid-albumin. 

{(a)  Proto-albumose        f  ^^^  P^^^^^-^  albumoses,  i.e 

2.  Proteoses  (6)  Hetero-albumose  those  which  are   formed 

1  ^  ^  I     first. 

\{c)  Deutero-albumose 

3.  Peptone. 

1.  Acid  albumin. — The  properties  of  the  infra-proteins  which  are 
the  first  degradation  products  in  the  cleavage  of  the  proteins  which 
occurs  during  digestion  have  been  described  in  Lesson  V.  (see  pp.  29 
and  48).  We  shall  find  later  that,  in  pancreatic  digestion,  alkali- 
albumin  is  formed  instead  of  acid-albumin.  The  theory  has  been 
put  forward  that  a  protein  is  capable  of  playing  the  part  of  a  base  in 


76  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

virtue   of  its   NHg  groups,   and   also   of   an   acid   in   virtue   of   its 
COOH  groups. 

2.  Proteoses.  —  These  are  the  intermediate  products  in  the 
hydrolysis  of  native  proteins  into  peptones. 

They  are  not  coagulated  by  heat ;  they  are  precipitated  but  not 
coagulated  by  alcohol ;  like  peptone  they  give  the  biuret  reaction. 
They  are  precipitated  by  nitric  acid,  the  precipitate  being  soluble  on 
heating,  and  reappearing  when  the  liquid  cools.  The  last  is  a 
distinctive  property  of  proteoses.     They  are  slightly  diffusible. 

The  difference  between  the  different  proteoses  is  mainly  one  of 
solubihty.  The  primary  proteoses  (proto-  and  hetero-)  are  precipitated 
by  saturation  with  magnesium  sulphate  or  sodium  chloride.  Deutero- 
proteose  is  not ;  it  is,  however,  precipitated  by  saturation  with 
ammonium  sulphate.  Proto-  and  deutero-  proteoses  are  soluble  in 
water :  hetero-proteose  is  not :  it  requires  a  salt  to  hold  it  in 
solution. 

3.  Peptones. — These  are  the  final  products  of  the  action  of  gastric 
juice  on  native  proteins.  If  the  action  is  very  prolonged,  polypeptides 
and  amino-acids  are  split  off  from  the  peptones,  but  in  the  usual 
short  stay  of  food  in  the  stomach  very  little  of  these  ultimate  cleavage 
products  is  found  there. 

They  are  soluble  in  water,  are  not  coagulated  by  heat,  and  are 
not  precipitated  by  nitric  a^cid,  copper  sulphate,  ammonium  sulphate, 
and  a  number  of  other  precipitants  of  proteins.  They  are  precipi- 
tated but  not  coagulated  by  alcohol.  They  are  also  precipitated  by 
tannin,  picric  acid,  potassio-mercuric  iodide,  phospho-molybdic  acid, 
and  phospho-tungstic  acid.  They  give  the  biuret  reaction  (rose-red, 
with  a  trace  of  copper  sulphate  and  caustic  potash  or  soda)  and  are 
readily  diffusible  through  animal  membranes. 

To  sum  up  :  the  main  action  of  the  gastric  juice  is  upon  the  pro- 
teins of  the  food,  converting  them  into  more  soluble  and  diffusible 
products.  The  protein  envelopes  of  the  fat  globules  are  dissolved,  and 
the  solid  fats  are  melted.  According  to  some,  gastric  juice  contains 
a  fat-spliting  ferment  in  small  quantities  which  acts  like  the  steapsin 
of  pancreatic  juice  ;  but  this  action  if  present  is  very  slight.  Starch 
is  unaffected ;  but  cane  sugar  is  inverted.  The  inversion  of  cane 
sugar  is  due  to  the  hydrochloric  acid  of  the  juice,  and  is  frequently 
assisted  by  inverting  ferments  contained  in  the  vegetable  food 
swallowed.  The  stomach  does  not  digest  itself,  because  it  forms  an 
antipepsin  ;  similarly  in  the  intestine  an  antitrypsin  is  formed.  The 
formation  of  anti-bodies  will  be  treated  under  the  heading  Immunity. 
(See  Blood.) 


THE   DIGESTIVE  JUICES 


77 


The  following  table  gives  us  at  a  glance  the  chief  characters  of 
proteoses  and  peptones  in  contrast  with  those  of  such  native  proteins 
as  albumin  and  globulin. 


Variety  of 
protein 

Action  of 
heat 

Action  of 
alcohol 

Action  of 
nitric  acid 

Action  of 

ammonium 

sulphate 

Action  of 

copper 

sulphate 

and  caustic 
potash 

! 

DiflEusi- 
bility 

Ditto 

1 

Slight 
Great 

1 

Albumin 

Globulin 

Proteoses 
Peptones 

Coagulated 
Ditto 

Not     coagu- 
lated 

Not     coagu- 
lated 

Precipitated, 
then  coagu- 
lated 

Ditto   - 

Precipitated 
but  not  co- 
agulated 

Precipitated 
but  not  co- 
agulated 

Precipitated 
in  the  cold ; 
not  readily 
soluble     on 
heating 

Ditto 

Precipitated 
in  the  cold ; 
readily    so- 
luble       on 
heating ; 
the  precipi- 
tate    reap- 
pears      on 
cooling  ^ 

Not     precipi- 
tated 

Precipitated 
by  complete 
saturation 

Precipitated 
by  half  satu- 
ration; also 
precipitated 
by  satura- 
tion with 
MgSO, 

Precipitated 
by  saturation 

Not       precipi- 
tated 

Violet 
colour 

Ditto 

Rose-red 
colour 
(biuret 
reaction) 

Rose-red 
colour 
(biuret 
reaction) 

'  In  the  case  of  deutero-albumose  this  reaction  only  occurs  in  the  presence  of 
excess  of  salt. 


LESSON  VIII 

THE   DIGESTIVE   JUICES   [continued) 

Pancreatic  Digestion 

1.  A  1-per-cent.  solution  of  sodium  carbonate,  to  which  a  little  glycerin 
extract  of  pancreas  ^  has  been  added,  forms  a  good  artificial  pancreatic  fluid. 

2.  Half  fill  three  test-tubes  with  this  solution. 

A.  To  this  add  half  its  bulk  of  diluted  egg-white  (1  in  10). 

B.  To  this  add  a  piece  of  fibrin. 

C.  Boil  this  ;  cool ;  then  add  fibrin. 

3.  Put  all  into  the  water-bath  at  40°  C.  After  half  an  hour,  test  A  and  B 
for  alkali-albumin  by  neutralisation,  for  proteoses  by  nitric  acid,  and  for 
proteoses  and  peptone  by  the  biuret  reaction. 

4.  Note  in  B  that  the  fibrin  does  not  swell  up  and  dissolve,  as  in  gastric 
digestion,  but  that  it  is  eaten  away  from  the  edges  to  the  interior. 

5.  In  C  no  digestion  occurs,  as  the  ferments  have  been  destroyed  by 
boiling, 

6.  Take  a  solution  of  starch,  equal  quantities  in  three  test-tubes. 

D.  To  this  add  a  few  drops  of  glycerin  extract  of  pancreas  (without 

the  sodium  carbonate), 

E.  To  this  add  a  few  drops  of  bile. 

F.  To  this  add  both  bile  and  pancreatic  extract. 

7.  Put  these  into  the  water  bath,  and  test  small  portions  of  each  every 
half-minute  by  the  iodine  reaction.  It  disappears  first  in  F ;  then  in  D  ; 
while  E  undergoes  no  change.  Test  D  and  F  for  maltose  by  Fehling's 
solution. 

8.  Shake  up  a  few  drops  of  olive  oil  with  artificial  pancreatic  juice 
(extract  of  pancreas  and  sodium  carbonate).  A  milky  fluid  (emulsion)  is 
formed,  from  which  the  oil  does  not  readily  separate  on  standing. 

9.  The  foregoing  experiments  illustrate  the  action  that  pancreatic  juice 
has  on  all  three  classes  of  organic  food. 

i.  On  Proteins. — Fibrin,  albumin,  &c.  are  converted  into  proteoses  and 
peptone  by  the  ferment  trypsin  in  an  alkaline  medium. 

ii.  On  Carholiydrates. — Starch  is  converted  into  sugar  (maltose)  by  the 
ferment  amylopsin,  especially  in  presence  of  bile. 

iii.  On  Fats. — These  are  emulsified.  In  the  body  they  are  also  split  into 
fatty  acid  and  glycerin  by  the  ferment  steapsin  ;  but  this  cannot  be  shown 
with  the  artificial  juice,  as  steapsin  is  not  soluble  in  glycerin. 

Bile 

1.  Ox  bile  is  given  round.  Observe  its  colour,  taste,  smell,  and  reaction 
to  litmus  paper. 

2.  Acidulate  a  little  bile  with  20-per-cent.  acetic  acid.    A  stringy  precipitate 

'  Benger's  liquor  pancreaticus  diluted  with  two  or  three  times  its  volume  of 
1-per-cent.  sodium  carbonate  may  be  used  instead. 


THE  DIGESTIVE  JUICES  79 

of  a  mucinoid  substance  is  obtained.     Filter  this  off  and  boil  the  filtrate  ;  no 
protein  coagiilable  by  heat  is  present. 

3.  Add  a  few  drops  of  bile  to  (a)  acid-albumin  prepared  as  described  in 
Lesson  V.,  and  (6)  solution  of  proteoses  to  which  half  its  volume  of  0'2.per. 
cent,  hydrochloric  acid  has  been  added.  A  precipitate  occurs  in  each  case. 
Bile  salts  precipitate  the  unpeptonised  protein  which  leaves  the  stomach. 

4.  Petteiiliofer'8  Test  for  Bile  Salts. — To  a  thin  film,  of  bile  in  a  capsule 
add  a  drop  of  solution  of  cane  sugar  and  a  drop  of  concentrated  sulphuric 
acid.  A  purple  colour  is  produced.  This  occurs  more  quickly  on  the 
application  of  heat.  The  test  may  also  be  performed  as  follows  : — Shake  up 
some  bile  and  cane  sugar  solution  in  a  test-tube  until  a  froth  is  formed. 
Pour  concentrated  sulphuric  acid  gently  down  the  side  of  the  tube  :  it  produces 
a  purple  colour  in  the  froth. 

5.  Gmelin's  Test  for  Bile  Pigments. — On  to  a  little  fuming  nitric  acid 
{i.e.  nitric  acid  containing  nitrous  acid  in  solution)  in  a  test-tube  pour  gently 
a  little  bile.  Notice  the  succession  of  colours — green,  blue,  red,  and  yellow 
— at  the  junction  of  the  two  liquids.  This  test  may  also  be  performed  in  a 
capsule.  Place  a  drop  of  fuming  nitric  acid  in  the  middle  of  a  thin  film  of 
bile  :  it  becomes  surrounded  by  rings  of  the  above-mentioned  colours. 

6.  Hay's  Test  for  Bile  Salts. — Take  two  beakers  full  of  water ;  to 
one  add  a  few  drops  of  bile,  or  solution  of  bile  salts.  Sprinkle  a  little 
flowers  of  sulphur  on  the  surface  of  each.  It  remains  floating  on  the 
pure  water ;  but  where  bile  is  present  the  surface  tension  of  the  water  is 
reduced,  and  the  sulphur  consequently  rapidly  sinks.  This  test  is  very 
sensitive. 

7.  Preparation  of  Glycocholic  Acid. — The  preparation  of  the  bile  acids 
is  usually  a  task  of  some  difficulty ;  the  following  exercise  is  a  simple  one, 
though  unfortunately,  for  reasons  which  are  not  explicable,  it  does  not  always 
succeed.  Take  a  stoppered  cylinder,  place  in  it  200  c.c.  of  ox  bile,  10  c.c.  of 
hydrochloric  acid,  and  25  c.c.  of  ether,  and  shake  vigorously.  Add  a  crystal 
of  glycocholic  acid,  and  allow  the  mixture  to  stand  in  a  cool  place.  In  a  time 
varying  from  a  few  minutes  to  some  hours,  a  mass  of  crystals  of  glycocholic 
acid  separates  out.  This  may  be  filtered  off,  washed,  and  dissolved  in  a  little 
boiling  water,  and  filtered  hot.  On  cooling,  needle-like  crystals  of  the  acid 
again  separate  out. 

8.  Cholesterin. — (a)  Examine  crystals  of  this  substance  with  the  micro- 
scope. Heat  these  on  a  slide  with  a  drop  of  sulphuric  acid  and  water  (5:1); 
the  edges  of  the  crystals  turn  red. 

(6)  Salkowski's  reaction.  Dissolve  some  cholesterin  in  chloroform  in  a 
dry  test-tube,  and  gently  shake  with  an  equal  amount  of  concentrated 
sulphuric  acid ;  the  solution  turns  red,  and  the  subjacent  acid  acquires 
a  green  fluorescence.  The  chloroformic  solution  of  cholesterin  is  rendered 
colourless  by  pouring  it  into  a  wet  test-tube,  and  the  colour  is  restored  by 
the  addition  of  sulphuric  acid. 

(c)  Liebermann's  reaction.  Two  or  three  drops  of  acetic  anhydride  are 
added  to  a  chloroformic  solution  of  cholesterin  and  then  sulphuric  acid  drop 
by  drop.  A  rose-red  colour  first  develops  ;  this  becomes  blue  and  finally 
bluish-green. 

THE  PANCREAS 

The  Pancreas  is  a  compound  tubulo-racemose  gland  ;  between 
the  secreting  acini  are  situated  little  masses  of  epithelial  cells  without 
ducts  called  *  islets  of  Langerhans.'  Examination  of  the  secreting 
cells  in  different  stages  of  activity  reveals  changes  comparable  to 
those  already  described   in   the  case  of   salivary  and   gastric   cells. 


80  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

Granules  indicating  the  presence  of  a  zymogen  which  is  called 
trypsinogen  (that  is,  the  precursor  of  trypsin,  the  most  important 
ferment  of  the  pancreatic  juice)  crowd  the  cells  before  secretion : 
these  are  discharged  during  secretion,  so  that  in  an  animal  whose 
pancreas  has  been  powerfully  stimulated  to  secrete,  as  by  the  ad- 
ministration of  pilocarpine,  the  granules  are  seen  only  at  the  free 
border  of  the  cells  (see  fig.  21). 

As  in  the  case  of  gastric  juice,  experiments  on  the  pancreatic 
secretion  are  usually  performed  with  an  artificial  juice,  made  by 
mixing  a  weak  alkaline  solution  (1-per-cent.  sodium  carbonate)  with 
an  extract  of  pancreas.  The  pancreas  should  be  kept  some  time 
before  the  extract  is  made,  so  as  to  ensure  that  the  transformation 
of  trypsinogen  into  trypsin  has  taken  place. 


Fig.  21.— Part  of  an  alveolus  of  the  rabbit's  pancreas  :  A,  before  discharge;  B,  after, 
(From  Foster,  after  KUhne  and  Lea.) 

Quantitative  analysis  of  human  pancreatic  juice  gives  the  following 
results : — 

Water        ....         97'6  per  cent. 
Organic  solids   ...  1*8       „ 

Inorganic  salts  ...  0*6       „ 

Dog's  pancreatic  juice  is  considerably  richer  in  solids. 
The  organic  substances  in  pancreatic  juice  are : — 

(a)  Ferments.     These  are  the  most  important  both  quantitatively 
and  functionally.     They  are  four  in  number  : — 

i.  Trypsin,  a  proteolytic  ferment. 

ii.  Amylopsin  or  pancreatic  diastase,  an  amylolytic  ferment. 

iii.  Steapsin,  a  fat-splitting  ferment. 

iv.  A  milk-curdling  ferment. 

(b)  A  small  amount  of  protein  matter,  coagulable  by  heat. 

(c)  Traces  of  leucine,  tyrosine,  xanthine,  and  soaps. 


THE  DIGESTIVE  JUICES  81 

The  inorganic  substances  in  pancreatic  juice  are  : — 
Sodium  chloride,  which  is  the  most  abundant,  and  smaller  quanti- 
ties of  potassium  chloride,  and  phosphates  of  sodium,  calcium,  and 
magnesium.     The  alkalinity  of  the  juice  is  due  to  phosphates  and 
carbonates,  especially  of  sodium. 


ACTION   OF   PANCREATIC   JUICE 

The  action  of  pancreatic  juice,  which  is  the  most  powerful  and 
important  of  all  the  digestive  juices,  may  be  described  under  the 
headings  of  its  four  ferments. 

1.  Action  of  Trypsin. — Trypsin  acts  like  pepsin,  but  with  certain 
differences,  which  are  as  follows  : — 

(a)  It  acts  in  an  alkaline,  pepsin  in  an  acid  medium. 

(b)  It  acts  more  rapidly  than  pepsin ;  deutero -proteoses  can  be 
detected  as  intermediate  products  in  the  formation  of  peptone. 
Primary  (i.e.  proto-  and  hetero-)  proteoses  have  not  been  found  ;  the 
action  is  apparently  too  rapid  to  admit  of  their  detection. 

(c)  The  first  degradation  product  is  alkali-albumin  in  place  of  the 
acid-albumin  of  gastric  digestion. 

(d)  It  acts  more  powerfully  on  certain  proteins  (such  as  elastin) 
which  are  difficult  of  digestion  in  gastric  juice.  Collagen,  however, 
is  not  digested. 

(e)  Acting  on  solid  proteins  like  fibrin,  it  eats  them  away  from 
the  surface  to  the  interior  ;  there  is  no  preliminary  swelling  as  in 
gastric  digestion. 

(/)  Trypsin  acts  further  than  pepsin,  decomposing  peptone  into 
simpler  products,  of  which  the  most  familiar  are  leucine  and  tyrosine. 

Besides  leucine  and  tyrosine,  other  amino-acids  such  as  aspartic 
acid,  glutamic  acid,  lysine,  arginine,  and  tryptophan  and  ammonia 
are  also  formed.  A  more  complete  list  of  the  cleavage  products  with 
their  chemical  constitution  is  given  on  pp.  31-35. 

We  know  that  the  action  of  proteolytic  enzymes  is,  by  the 
process  of  hydrolysis,  to  spHt  the  heavy  protein  molecule  into 
smaller  and  smaller  molecules ;  first  we  get  proteoses,  then  peptones, 
and  finally  after  prolonged  action  simple  substances  like  leucine  and 
tyrosine. 

This  cleavage  is  more  easily  performed  by  the  more  powerful 
tryptic  enzyme  than  by  the  comparatively  feeble  agent,  pepsin- 
hydrochloric  acid.  Still  we  have  already  seen  that  by  the  very  pro- 
longed action  of  the  latter,  leucine,  tyrosine,  and  other  amino-acids 

G 


82  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

are  formed,  although  the  quantity  of  these  in  an  ordinary  gastric 
digest  is  negUgible.  The  essential  difference  between  the  two 
enzymes  is  one  of  degree  only  ;  trypsin  is  by  far  the  more  powerful 
catalytic  agent,  and  increases  the  velocity  of  the  reaction  much  more 
markedly  than  does  pepsin-hydrochloric  acid. 

2.  Action  of  Amylopsin. — The  conversion  of  starch  into  maltose 
is  the  most  powerful  and  rapid  of  all  the  actions  of  the  pancreatic 
juice.  It  is  much  more  powerful  than  saliva,  and  will  act  even  on 
unboiled  starch.  The  absence  of  this  ferment  in  the  pancreatic  juice 
of  infants  is  an  indication  that  milk,  and  not  starch,  is  their  natural 
diet. 

3.  Action  on  Fats. — The  action  of  pancreatic  juice  on  fats  is  a 
double  one :  it  forms  an  emulsion,  and  it  decomposes  the  fats  into 
fatty  acids  and  glycerin  by  means  of  its  fat-splitting  ferment  steapsin. 
The  fatty  acids  unite  with  the  alkaline  bases  to  form  soaps  {saponifica- 
tio7i).     The  chemistry  of  this  is  described  on  p.  25. 

With  regard  to  the  formation  of  emulsions  the  following  are  the 
principal  facts.  If  olive  oil  and  water  are  shaken  up  together  and 
the  mixture  is  then  allowed  to  stand,  the  finely  divided  oil  globules 
soon  separate  from  and  float  on  the  surface  of  the  water ;  but  if  the 
oil  is  shaken  up  with  a  solution  of  soap,  the  conditions  of  surface 
tension  are  such  that  the  oil  globules  remain  as  such  in  the  mixture, 
and  a  milky  fluid  or  emulsion  is  the  result.  This  emulsion  is 
rendered  still  more  permanent  by  the  presence  of  colloid  or  viscous 
materials,  especially  when  a  small  amount  of  free  fatty  acid  is  being 
continually  liberated.  The  acid  combines  with  the  alkali  to  form 
a  soap  ;  the  soap  probably  forms  a  thin  layer  on  the  outside  of  each 
oil  globule,  which  prevents  them  running  together  again.  Pancreatic 
fluid  possesses  all  the  necessary  qualifications  for  forming  an 
emulsion.  It  is  alkaline,  and  it  liberates  fatty  acids  from  the  fat : 
these  acids  form  soap  with  the  alkali  present ;  moreover,  it  is  viscous 
from  the  presence  of  protein. 

4.  Milk-curdling  Ferment. — The  addition  of  pancreatic  extracts 
to  milk  causes  clotting,  which  differs  in  some  of  its  details  from  the 
curdling  produced  by  rennin  ;  but  this  action  can  hardly  ever  be 
called  into  play,  as  the  milk  upon  which  the  juice  has  to  act  has  been 
already  curdled  by  the  rennin  of  the  stomach. 


THE  DiaESTlVE  JUICES  83 

THE   SECRETION   OF  PANCREATIC   JUICE 

One  of  the  most  effective  ways  of  producing  a  flow  of  the  juice 
is  to  introduce  acid  into  the  duodenum,  and  no  doubt  the  acid  of  the 
gastric  juice  is  the  normal  stimulus  for  the  pancreatic  flow.  This  flow 
still  occurs  when  all  the  nerves  supplying  the  duodenum  and  pancreas 
are  cut,  and  it  was  held  by  Popielski  and  by  Wertheimer  and  Le 
Page  that  it  must  be  due  to  a  local  reflex,  the  centres  being  situated 
in  the  scattered  ganglia  of  the  pancreas  and  of  the  solar  plexus. 
Starling  and  Bayliss,  however,  pointed  out  that  it  cannot  be  a 
nervous  reflex,  since  it  occurs  after  extirpation  of  the  solar  plexus, 
and  destruction  of  all  nerves  passing  to  an  isolated  loop  of  intestine. 
Moreover,  atropine  does  not  paralyse  the  secretory  action.  It  must 
therefore  be  due  to  direct  excitation  of  the  pancreatic  cells  by  a 
substance  or  substances  conveyed  to  the  gland  from  the  bowel  by  the 
blood-stream. 

The  exciting  substance  is  not  acid  ;  injection  of  0*4  per  cent,  of 
hydrochloric  acid  into  the  blood-stream  has  no  influence  on  the 
pancreas.  The  substance  in  question  must  be  produced  in  the 
intestinal  mucous  membrane  under  the  influence  of  the  acid.  This 
conclusion  was  confirmed  by  experiment.  If  the  mucous  membrane 
of  the  duodenum  or  jejunum  is  exposed  to  the  action  of  04  per  cent, 
hydrochloric  acid,  a  body  is  produced  which,  when  injected  into  the 
blood-stream  in  minimal  doses,  produces  a  copious  secretion  of  pan- 
creatic juice.  This  substance  is  termed  secretm.  It  is  associated  with 
another  substance  which  lowers  arterial  blood-pressure.  The  two 
substances  are  not  identical,  since  acid  extracts  of  the  lower  end  of 
the  ileum  produce  a  lowering  of  blood-pressure,  but  have  no  excitatory 
influence  on  the  pancreas. 

Secretin  is  split  off  from  a  precursor,  prosecretin,  which  is  present 
in  relatively  large  amounts  in  the  duodenal  mucous  membrane,  and 
gradually  diminishes  in  amount  throughout  the  intestine  until  it 
entirely  disappears  in  the  ileum.  Pro-secretin  can  be  dissolved 
out  of  the  mucous  membrane  by  normal  saline  solution.  It  has  no 
influence  on  the  pancreatic  secretion.  Secretin  can  be  split  off  from 
it  by  boiling  or  by  treatment  with  acid. 

What  secretin  is  chemically,  we  do  not  yet  know.  It  is  soluble  in 
alcohol  and  ether.  It  is  not  a  protein,  but  probably  is  an  organic 
substance  of  low  molecular  weight.  It  is,  moreover,  the  same  sub- 
stance in  all  animals,  and  not  specific  to  different  kinds  of  animals. 

Whether  there  are  any  secretory  nerves  for  the  pancreas  is  at 
present  doubtful.     Pawlow  thought  he  had  discovered  them  in  the 

g2 


84  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

vagus ;  but  as  he  did  not  exclude  in  his  experiments  the  passage  of 
the  acid  chyme  from  the  stomach  into  the  duodenum,  it  is  probable 
that  the  pancreatic  secretion  he  obtained  was  due  to  that  circum- 
stance and  the  consequent  formation  of  secretin. 

Injection  of  secretin  also  stimulates  the  flow  of  bile. 

Secretin  is  an  instance  of  the  chemical  messengers  or  hormones 
(Starling)  of  the  body.  Evidence  is  accumulating  to  show  that 
hormones  are  extremely  important.  It  has  already,  for  instance, 
been  shown  that  one  called  gastrin  is  formed  as  the  result  of  salivary 
digestion,  and  stimulates  the  flow  of  gastric  juice.  Another  is 
formed  from  the  foetal  tissues,  which,  passing  into  the  mother's 
circulation,  stimulates  the  mammae  to  enlarge  and  secrete  milk. 

INTESTINAL    DIGESTION 

The  pancreatic  juice  does  not  act  alone  on  the  food  in  the  intes- 
tines. There  are,  in  addition,  the  bile,  the  succus  entericus  (secreted 
by  the  crypts  of  Lieberkiihn),  and  bacterial  action  to  be  considered. 

The  succus  entericus  or  intestinal  juice  has  no  action  on  fats  or 
native  proteins,  but  it  appears  to  have  to  some  extent  the  power  of 
converting  starch  into  sugar ;  its  best  known  action  is  due  to  a  ferment 
it  contains  called  invertin,  which  inverts  saccharoses — that  is,  con- 
verts cane  sugar  and  maltose  into  glucose.  The  original  use  of  the 
term  '  inversion '  has  been  explained  on  p.  17.  It  may  be  extended 
to  include  the  similar  hydrolysis  of  other  saccharoses,  although  there 
may  be  no  formation  of  levo-rotatory  substances.  There  are  probably 
several  inverting  ferments  in  the  succus  entericus,  one  of  which  acts 
on  cane  suger,  one  on  maltose,  and  one  on  milk  sugar. 

A  few  years  ago,  however,  Pawlow  showed  that  succus  entericus 
has  a  still  more  important  action,  which  is  to  intensify  the  proteo- 
lytic power  of  the  pancreatic  juice.  Fresh  pancreatic  juice  has 
very  little  power  on  proteins,  for  what  it  contains  is  not  trypsin, 
but  its  precursor,  trypsinogen. 

If  fresh  pancreatic  and  intestinal  juices  are  mixed  together,  the 
result  is  a  very  powerful  proteolytic  mixture,  though  neither  juice  by 
itself  has  any  proteolytic  activity.  The  substance  in  the  intestinal 
juice  that  activates  trypsinogen  or,  in  other  words,  liberates  trypsin 
from  trypsinogen  has  been  called  by  Pawlow  a  '  ferment  of  the 
ferments,'  or  enter o-kinase. 

Intestinal  juice  contains  another  ferment  called  ercpsin  (Otto 
Cohnheim),  which  is  capable  of  breaking  up  proteoses  and  peptone 
into  simple  substances  (leucine,  tyrosine,  hexone  bases,  ammonia, 
&c.),  and  so  assisting  the  action  of  trypsin. 


THE   DIGESTIVE   JUICES  85 

Bacterial  Action. — The  gastric  juice  is  an  antiseptic ;  the  pan- 
creatic juice  is  not.  An  alkahne  fluid  like  pancreatic  juice  is  just 
the  most  suitable  medium  for  bacteria  to  flourish  in.  Even  in 
an  artificial  digestion  the  fluid  is  very  soon  putrid,  unless  special 
precautions  to  exclude  or  kill  bacteria  are  taken.  It  is  often  difficult 
to  say  where  pancreatic  action  ends  and  bacterial  action  begins,  as 
many  of  the  bacteria  that  grow  in  the  intestinal  contents,  having 
reached  that  situation  in  spite  of  the  gastric  juice,  act  in  the  same 
way  as  the  pancreatic  juice.  Some  form  sugar  from  starch,  others 
peptone,  leucine,  and  tyrosine  from  proteins,  while  others  again 
break  up  fats.  There  are,  however,  certain  actions  that  are  entirely 
or  mainly  due  to  these  putrefactive  organisms. 

i.  On  carbohydrates.  The  most  frequent  fermentation  they  set 
up  is  the  lactic  acid  fermentation :  this  may  go  further  and  result  in 
the  formation  of  carbonic  acid,  hydrogen,  and  butyric  acid  (see  p.  19). 
Cellulose  is  broken  up  into  carbonic  acid  and  methane.  This  is  the 
chief  cause  of  the  gases  in  the  intestine,  the  amount  of  which  is 
increased  by  vegetable  food. 

ii.  On  fats.  In  addition  to  acting  like  steapsin,  lower  acids 
(valeric,  butyric,  &c.)  are  produced.  The  formation  of  acid  products 
from  fats  and  carbohydrates  gives  to  the  intestinal  contents  an  acid 
reaction.  Eecent  researches  show  that  the  contents  become  acid 
much  higher  up  in  the  small  intestine  than  was  formerly  considered 
to  be  the  case.  These  organic  acids  do  not  hinder  pancreatic  diges- 
tion to  any  appreciable  extent. 

iii.  On  proteins.  Fatty  acids  and  amino-acids  are  produced,  but 
these  putrefactive  organisms  are  specially  efficacious  in  liberating  the 
protein  cleavage  products  which  have  an  evil  odour  like  indole, 
skatole,  and  phenol.     There  are  also  gaseous  products  in  some  cases. 

If  excessive,  putrefactive  processes  are  harmful ;  if  within  normal 
limits,  they  are  useful,  helping  the  pancreatic  juice  and,  further,  pre- 
venting the  entrance  into  the  body  of  poisonous  products.  It  is 
possible  that,  in  digestion,  poisonous  alkaloids  are  formed.  Certainly 
this  is  so  in  one  well-known  case.  Lecithin,  a  material  contained  in 
small  quantities  in  many  foods,  and  in  large  quantities  in  egg-yolk 
and  brain,  is  broken  up  by  the  pancreatic  juice  into  glycerin,  phos- 
phoric acid,  fattj^  acids,  and  an  alkaloid  called  choline.  We  are, 
however,  protected  from  the  poisonous  action  of  choline  by  the 
bacteria,  which  break  it  up  into  carbonic  acid,  methane,  and 
ammonia. 


86 


ESSENTIALS   OF  CHEMICAL  PHySIGLOGY 


LEUCINE  AND  TYROSINE 

These  two  substances  have  been  frequently  mentioned  in  the 
preceding  pages,  and  they  are  the  final  cleavage  products  of  proteins 
which  have  been  longest  known.  Leuciiie  is  usually  the  most 
abundant  of  all  the  cleavage  products  (see  table  on  p.  35). 

We  have  already  learnt  that  they  are  amino-acids,  and  that 
leucine  is  amino-caproic  acid  (see  p.  31).  There  are,  however,  several 
isomeric  amino-caproic  acids.     It  was  thought  until  quite  recently 


Fig.  22. — Leucine  crystals. 


Fig.  23.— Tyrosine  crystals 


that  leucine  was  the  amino-acid  of  normal  caproic  acid,  but  it  has 
been  shown  to  be  a-amino-iso-butyl-acetic  acid.  The  difference  in  the 
structure  of  these  two  compounds  may  be  represented  by  the  following 
graphic  formulae  : — 

/CH3 

CH., 

CH2  Iso-butyl 

CH, 


Normal 


a-amino-caproic 
acid 


CH3CH3 


2  a-ammo- 

CH.NH2  acetic  acid 

ICOOH 


CH 
CH, 
CH.NH2 
COOH 


Tyrosine  is  a  little  more  complicated,  as  it  is  not  only  an  amino-acid, 
but  also  contains  an  aromatic  radical  (see  p.  34).  Figs.  22  and  23 
represent  the  crystalline  forms  of  leucine  and  tyrosine. 


THE   DIGESTIVE  JUICES  87 


EXTIRPATION   OF  THE  PANCREAS 

Complete  removal  of  the  pancreas  in  animals  and  diseases  of 
the  pancreas  in  man  produce  a  condition  of  diabetes,  in  addition 
to  the  loss  of  pancreatic  action  in  the  intestines.  Grafting  the 
pancreas  from  another  animal  into  the  abdomen  of  the  animal 
from  which  the  pancreas  has  been  previously  removed  relieves  the 
diabetic  condition. 

How  the  pancreas  acts  otherwise  than  in  producing  the  pancreatic 
juice  is  not  known.  It  must,  however,  have  other  functions  related 
to  the  general  metabolic  phenomena  of  the  body,  which  are  disturbed 
by  removal  or  disease  of  the  gland.  This  is  an  illustration  of  a 
universal  truth — viz.  that  each  part  of  the  body  does  not  merely  do 
its  own  special  work,  but  is  concerned  in  the  great  cycle  of  changes 
which  is  called  general  metabolism.  Interference  with  any  organ 
upsets,  not  only  its  specific  function,  but  causes  disturbances  through 
the  body  generally.  The  interdependence  of  the  circulatory  and 
respiratory  systems  is  a  well-known  instance.  Eemoval  of  the 
thyroid  gland  upsets  the  whole  body,  producing  widespread  changes 
known  as  myxoedema.  Eemoval  of  the  testes  produces,  not  only  a 
loss  of  the  spermatic  secretion,  but  changes  the  whole  growth  and 
appearance  of  the  animal.  Eemoval  of  the  greater  part  of  the 
kidneys  produces  rapid  wasting  and  the  breaking  down  of  the  tissues 
to  form  an  increased  quantity  of  urea.  The  precise  way  in  which 
these  glands  are  related  to  the  general  body  processes  is,  however,  a 
subject  of  which  we  know  as  yet  very  little.  The  theory  at  present 
most  in  favour  is  that  certain  glands  produce  an  internal  secretion, 
which  leaves  the  gland  via  the  lymph  or  venous  blood,  and  is  then 
distributed  to  minister  to  parts  elsewhere.  Eemoval  of  such  glands 
as  the  thyroid  or  suprarenal  produces  disease  or  death  because  this 
internal  secretion  can  no  longer  be  formed.  In  the  case  of  the 
pancreas,  the  external  secretion  of  the  pancreas  (that  is,  pancreatic 
juice)  is  formed  by  the  cells  lining  the  acini,  and  the  internal  secre- 
tion, stoppage  of  which  in  some  way  leads  to  diabetes,  has  been 
attributed  by  some  to  the  islets  of  Langerhans ;  but  if  these  islets 
are  only  stages  in  the  formation  of  acini,  as  they  have  been  shown 
to  be,  it  is  difficult  to  fully  accept  this  view. 

In  diabetes  the  oxidative  powers  of  the  body  cells  are  lessened,  and 
the  capability  of  these  cells  to  prepare  sugar  for  oxidation  is  impaired. 
In  this  process  the  sugar  or  its  derivative  glycuronic  acid  is  split  into 
smaller  molecules,  and  ultimately   into   water   and   carbon    dioxide.     The 


88  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

close  relationship  of  sugar  and  glycuronic  acid  is  shown  by  the  following 

formulae : — 

COH  COH 

(CHOH),  (CHOH)^ 

CH^OH  COOH 

[dextrose]  [glycuronic  acid] 

That  is,  two  hydrogen  atoms  in  the  CH2OH  group  are  replaced  by  one  of 
oxygen.  This  oxidation  is  readily  brought  about  in  the  body,  and  glycuronic 
acid  is  usually  found  in  diabetic  urine  ;  but  the  further  oxidation  into  water 
and  carbon  dioxide  is  a  more  difficult  task,  because  it  involves  the  disruption 
of  the  linkage  of  the  carbon  atoms.  Perhaps  it  is  here  that  the  internal 
secretion  of  the  pancreas  is  effective.  This,  however,  is  at  present  a  mere 
theory,  and  certainly  Lepine's  idea  that  the  ferment  of  the  pancreatic 
internal  secretion  is  one  which  initiates  glycolysis  or  sugar-splitting  in  the 
blood,  has  been  abundantly  disproved.  It  may  be  that  the  active  principle 
of  the  pancreatic  internal  secretion  stimulates  the  glycolytic  action  of  the 
tissue-cells.  It  is  conceivable  that  in  the  other  great  cause  of  experimental 
glycosuria,  namely,  injury  to  nervous  structures,  as  in  Bernard's  puncture 
experiment,  the  derangement  of  the  nervous  system  exerts  some  unknown 
influence  on  the  pancreas  as  well  as  on  the  liver. 

Many  poisons  produce  temporary  glycosuria,  but  the  most  interesting 
and  powerful  of  these  is  phloridzin.  The  diabetes  produced  is  very  intense. 
Phloridzin  is  a  glucoside,  but  the  sugar  passed  in  the  urine  is  too  great  to  be 
accounted  for  by  the  small  amount  of  sugar  derivable  from  the  drug. 
Besides  that,  phloretin,  a  derivative  of  phloridzin,  free  from  sugar,  produces 
the  same  results. 

Phloridzin  produces  diabetes  in  starved  animals,  or  in  those  in  which  any 
carbohydrate  store  must  have  been  got  rid  of  by  the  previous  administration 
of  the  same  drug.  Phloridzin-diabetes  is  therefore  analogous  to  those  intense 
forms  of  diabetes  in  man  in  which  the  sugar  must  be  derived  from  proto- 
plasmic metabolism. 

A  puzzling  feature  is  the  absence  of  an  increase  of  sugar  in  the  blood  ;  if 
the  phloridzin  is  directly  injected  into  one  renal  artery,  sugar  rapidly  appears 
in  the  secretion  of  that  kidney  ;  the  sugar  is  formed  within  the  kidney  cells 
from  some  substance  in  the  blood,  but  whether  that  substance  is  protein  or 
not  is  uncertain.  The  action  of  the  kidney  cells  in  forming  sugar  has  been 
compared  to  that  of  the  mammary  cells  in  forming  lactose. 

Death  in  diabetic  patients  is  usually  preceded  by  deep  coma,  or  uncon- 
sciousness. Some  poison  must  be  produced  that  acts  soporifically  upon  the 
brain.  The  breath  and  urine  of  these  patients  smell  strongly  of  acetone ; 
hence  the  term  acetoncBmia.  This  apple-like  smell  should  always  suggest 
the  possible  onset  of  coma  and  death,  but  it  is  quite  certain  that  acetone 
(which  can  certainly  be  detected  in  the  urine)  is  not  the  true  poison ; 
ethyl-diacetic  acid,  which  accompanies  and  is  the  source  of  the  acetone,  was 
regarded  by  some  as  the  actual  poison ;  but  these  substances,  when  intro- 
duced into  the  circulation  artificially,  do  not  cause  serious  symptoms.  The 
principal  poison  is  /S-hydroxybutyric  acid  or  its  amino  derivative.  The 
alkalinity  and  carbonic  acid  of  the  blood  are  decreased,  and  the  ammonia 
of  the  urine  is  increased :  this  indicates  an  attempt  of  the  body  to  neutralise 
the  poisonous  acid.  The  acid  is  the  source  of  the  ethyl-diacetic  acid  and 
of  the  acetone.  Research  has  shown  that  /3-hydroxybutyric  acid  originates 
in  the  body  from  fat. 


THE   DIGESTIVE  JUICES  89 

THE  BILE 

Bile  is  the  secretion  of  the  liver  which  is  poured  into  the  duo- 
denum :  it  has  been  collected  in  living  animals  by  means  of  a 
biliary  fistula  ;  the  same  operation  has  occasionally  been  performed 
in  human  beings.  At  death  the  gall  bladder  yields  a  good  supply 
of  bile  which  is  more  concentrated  than  that  obtained  from  a 
fistula. 

Bile  is  being  continuously  poured  into  the  intestine,  but  there 
is  an  increased  discharge  immediately  on  the  arrival  of  food  in 
the  duodenum ;  there  i-s  a  second  increase  in  secretion  a  few  hours 
later. 

Though  the  chief  blood  supply  of  the  liver  is  by  a  vein  (the  portal 
vein),  the  amount  of  blood  in  the  liver  varies  with  its  needs,  being 
increased  during  the  periods  of  digestion.     This  is  due  to  the  fact 
that  in  the  area  from  which  the  portal 
vein  collects  blood — stomach,  intestine,  #    v    ^f 

spleen,  and  pancreas — the  arterioles  are  ^  i^    ^?  ^^  ^A 

all  dilated,  and  the  capillaries  are  thus     M  ^m  "^  \''    ♦     [^ 
gorged  with  blood.     Further,  the  active       ^^  ^  9  ^K  ^"J^ 
peristalsis    of     the    intestine    and    the  , '  M     ^     ^^ 

pumping  action   of   the  spleen   are  ad-  ^  ^       ^  K^^' 

ditional   factors   in  driving  more  blood  j;^,^^M? 

onwards  to  the  hver.  ^^HH^^^*^''" 

The  bile  is  secreted  from  the  portal         ^  _ 

^  Fig.  24.— HaBoiatoidin  crystals. 

blood  at  a  much  lower   pressure    than 

one  finds  in  glands,  such  as  the  salivary  glands,  the  blood  supply 
of  which  is  arterial.  Heidenhain  found  that  the  pressure  in  the  bile 
duct  of  a  dog  averaged  15  mm.  of  mercury,  which  is  about  double 
that  in  the  portal  vein. 

The  second  increase  in  the  flow  of  bile — that  which  occurs  some 
hours  after  the  arrival  of  the  semi-digested  food  (chyme)  in  the 
intestine — was  at  one  time  attributed  to  the  effect  of  the  digestive 
products  carried  by  the  blood  to  the  liver  stimulating  the  hepatic 
cells  to  activity:  this  was  supported  by  the  fact  that  protein  food 
increases  the  quantity  of  bile  secreted,  whereas  fatty  food,  which 
is  absorbed,  not  by  the  portal  vein,  but  by  the  lacteals,  has  no 
such  effect.  The  facts  are  now  more  readily  explained  by  the 
circumstance  that  secretin  is  a  stimulant  of  the  liver  as  well  as  of 
the  pancreas. 

The  chemical  processes  by  which  the  constituents  of  the  bile  are 
formed  are  obscure.     We,  however,  know  that  the  biliary  pigment  is 


90  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

produced  by  the  decomposition  of  haBmoglobin.  Bilirubin  is,  in  fact, 
identical  with  the  iron-free  derivative  of  haemoglobin  called  haemat- 
oidin,  which  is  found  in  the  form  of  crystals  in  old  blood-clots  such 
as  occur  in  the  brain  after  cerebral  haemorrhage  (see  fig.  24). 

An  injection  of  haemoglobin  into  the  portal  vein,  or  of  substances 
such  as  water  which  liberate  haemoglobin  from  the  red  blood  corpuscles, 
produces  an  increase  of  bile  pigment.  If  the  spleen  takes  any  part 
in  the  elaboration  of  bile  pigment,  it  does  not  proceed  so  far  as  to 
hberate  haemoglobin  from  the  corpuscles.  No  free  haemoglobin  is 
discoverable  in  the  blood  plasma  in  the  splenic  vein. 

The  amount  of  bile  secreted  is  differently  estimated  by  different 
observers  ;  the  amount  secreted  daily  in  man  appears  to  vary  from 
500  c.c.  to  1  litre  (1,000  c.c). 

THE   CONSTITUENTS   OF   BILE 

The  constituents  of  the  bile  are  the  bile  salts  proper  (taurocholate 
and  glycocholate  of  soda),  the  bile  pigments  (bilirubin,  biliverdin),  a 
mucinoid  substance,  small  quantities  of  fats,  soaps,  cholesterin, 
lecithin,  urea,  and  mineral  salts,  of  which  sodium  chloride  and  the 
phosphates  of  iron,  calcium,  and  magnesium  are  the  most  im- 
portant. 

Bile  is  a  yellowish,  reddish-brown  or  green  fluid,  according  to 
to  the  relative  preponderance  of  its  two  chief  pigments.  It  has  a 
musk-like  odour,  a  bitter-sweet  taste,  and  a  neutral  or  faintly  alkaline 
reaction. 

The  specific  gravity  of  human  bile  from  the  gall  bladder  is 
1026  to  1032;  that  from  a  fistula,  1010  to  1011.  The  greater  con- 
centration of  gall-bladder  bile  is  partly  but  not  wholly  explained 
by  the  addition  to  it  from  the  walls  of  that  cavity  of  the  mucinoid 
material. 

The  amount  of  solids  in  gall-bladder  bile  varies  from  9  to  14  per 
cent.,  in  fistula  bile  from  1-5  to  3  per  cent.  The  following  table 
shows  that  this  low  percentage  of  solids  is  almost  entirely  due  to  want 
of  bile  salts.  This  can  be  accounted  for  in  the  way  first  suggested 
by  Schiff — that  there  is  normally  a  bile  circulation  going  on  in  the 
body ;  a  large  quantity  of  the  bile  salts  that  passes  into  the  intestine 
is  first  split  up,  then  reabsorbed  and  again  secreted.  Such  a 
circulation  would  obviously  be  impossible  in  cases  where  all  the  bile 
is  discharged  to  the  exterior. 

The  following  table  gives  some  important  analyses  of  human 
bile :— 


THE   DIGESTIVE  JUICES 


91 


Constituents 

Fistula  bile 

(healtliy  woman. 

Copeman  and 

Winston) 

Fistula  bile  (case 

of  cancer.   Yeo  and 

Herroun) 

Normal  bile 
(Prerichs) 

I       9-14 
'      .1-18 

2-98 
0-78 

14-08 
85-92 

Sodium  g^lycocholate 
Sodium  taurocholate 
Cholesterin,  lecithin,  fat 
Mucinoid  material  . 
Pigment .... 
Inorganic  salts 

Total  solids      . 
Water  (by  difference) 

0-6280        1 

0-0990 
0-1725 
0-0725 
0-4510 

0-165 

0-055 
0-038 

1         0-148 
0-878 

1-284 
98-716 

1-4230 
98-5570 

100-0000 

100-000 

100-00 

, 

Bile  Mucin. — There  has  been  considerable  diversity  of  opinion  as 
to  whether  bile  mucin  is  really  mucin.  The  most  recent  work  in 
Hammarsten's  laboratory  shows  that  differences  occur  in  different 
animals.  Thus  in  the  ox  there  is  very  little  true  mucin,  but  a  great 
amount  of  nucleo-protein ;  in  human  bile,  on  the  other  hand,  there  is 
very  little  if  any  nucleo-protein  ;  the  mucinoid  material  present  there 
is  really  mucin.  (On  the  general  characters  of  Mucin  and  Nucleo- 
PROTEiNS  see  pp.  45  to  47.) 

The  Bile  Salts. — The  bile  contains  the  sodium  salts  of  complex 
amino-acids  called  the  bile  acids.  The  acids  most  frequently  found 
are  glycocholic  and  taurocholic  acids.  The  former  is  more  abundant 
in  the  bile  of  man  and  herbivora ;  the  latter  in  carnivora  like  the 
dog.  The  most  important  difference  between  the  two  acids  is  that 
taurocholic  acid  contains  sulphur,  and  glycocholic  acid  does  not. 

Glycocholic  acid  (C26H43NO6)  is  by  the  action  of  dilute  acids  and 
alkalis,  and  also  in  the  intestine,  hydrolysed  and  split  into  glycine  or 
amino-acetic  acid  and  cholalic  acid. 


Cor.Hj 


'2G 


3NO6  +  H2O: 


:CH2.NH2.COOH+C24H4o05 

[glycocholic  acid]  [glycine]  [cholalic  acid] 

The  glycocholate  of  soda  has  the  formula  C26H42NaN06. 
Taurocholic  acid  (O2GH45NO7S)  similarly  splits  into  taurine   or 
amino-ethyl-sulphonic  acid  and  cholalic  acid, 

C26H45NO7S  +  H,0=C2H4.NH2.HS03  4-  C24H40O5 

[taurocholic  acid]  [taurine]  [cholalic  acid] 

The  taurocholate  of  soda  has  the  formula  C26H44NaN07S. 
Glycocholic   and   taurocholic   acids    have   lately   been   prepared 
synthetically  from  cholalic  acid  and  glycine  and  taurine  respectively. 
The  colour  reaction  called  Pettenkofer's  reaction,  described  in  the 


92  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

practical  exercises  at  the  head  of  this  lesson,  is  due  to  the  presence 
of  cholalic  acid.  The  sulphuric  acid  acting  on  sugar  forms  a  small 
quantity  of  furfuraldehyde,  in  addition  to  other  products.  It  is  the 
furfuraldehyde  which  gives  the  purple  colour  with  cholalic  acid. 

The  Bile  Pigments.— The  two  chief  bile  pigments  are  bilirubin 
and  biliverdin.  Bile  which  contains  chiefly  the  former  (such  as  dog's 
bile)  is  of  a  golden  or  orange-yellow  colour,  while  the  bile  of  many 
herbivora,  which  contains  chiefly  biliverdin,  is  either  green  or  bluish 
green.  Human  bile  is  generally  described  as  containing  chiefly  bili- 
rubin, but  there  have  been  some  cases  described  in  which  biliverdin 
was  in  excess.  The  bile  pigments  show  no  absorption  bands  with  the 
spectroscope ;  their  origin  from  the  blood  pigment  has  already  been 
stated  (p.  90). 

Bilirubin  has  the  formula  CigHigNgOa:  it  is  thus  an  iron-free 
derivative  of  haemoglobin.  The  iron  is  apparently  stored  up  in  the 
liver  cells,  perhaps  for  future  use  in  the  manufacture  of  new  hasmo- 
globin.     The  bile  contains  only  a  trace  of  iron. 

Biliverdin  has  the  formula  Ci6H,gN204  {i.e.  one  atom  of  oxygen 
more  than  in  bilirubin) :  it  may  occur  as  such  in  bile ;  it  may  be 
formed  by  simply  exposing  red  bile  to  the  oxidising  action  of  the 
atmosphere ;  or  it  may  be  formed,  as  in  Gmelin's  test,  by  the  more 
vigorous  oxidation  produced  by  fuming  nitric  acid. 

Gmelin's  test  consists  of  a  play  of  colours — green,  blue,  red,  and 
finally  yellow — produced  by  the  oxidising  action  of  fuming  nitric  acid 
(that  is,  nitric  acid  containing  nitrous  acid  in  solution).  The  end  or 
yellow  product  is  called  choletelin,  CigHigNsOe. 

Hydrobilirubin. — If  a  solution  of  bilirubin  or  biliverdin  in  dilute 
alkali  is  treated  with  sodium  amalgam  or  allowed  to  putrefy,  a  brown- 
ish pigment  is  formed  called  hydrobilirubin,  C32H44N4O7.  With  the 
spectroscope  it  shows  a  dark  absorption  band  between  b  and  F,  and  a 
fainter  band  in  the  region  of  the  D  line. 

Urobilin. — Hydrobilirubin  is  interesting  because  a  similar  substance 
is  formed  from  the  bile  pigment  by  reduction  processes  in  the  intestine, 
and  constitutes  stercohilin,  the  pigment  of  the  faeces.  Some  of  this 
is  absorbed  and  ultimately  leaves  the  body  in  the  urine  as  one  of  its 
pigments  called  urobilin.  A  small  quantity  of  urobilin  is  sometimes 
found  preformed  in  the  bile.  The  identity  of  urobilin  and  stercohilin 
has  been  frequently  disputed,  but  the  recent  work  of  Garrod  and 
Hopkins  has  confirmed  the  old  statement  that  they  are  the  same 
substance  with  different  names.  Urobilin  has  a  well-marked  absorption 
band  in  the  region  of  the  F  line,  and  when  partially  precipitated  from 
an  alkaline  solution  by  acidification,  it  also  shows  an  absorption  band 


THE   DIGESTIVE  JUICES 


93 


in  the  region  of  the  E  line.  Hydrobilirubin  differs  from  urobilin  in 
containing  much  more  nitrogen  in  its  molecule  (9*2  instead  of  4*1  per 
cent.),  and  is  probably  a  product  of  less  complete  reduction  than 
urobilin.  (See  further  Lesson  XXVI.)  Urobilin  is  also  formed,  by 
the  oxidation  of  hsemopyrrol  (see  Haemoglobin,  p.  114). 

Cholesterin. — This  substance  is  contained,  not  only  in  bile,  but 
very  largely  in  nervous  tissues.  Like  lecithin,  it  is  an  abundant 
constituent  of  the  white  substance  of 
Schwann.  It  is  found  also  in  blood  cor- 
puscles, and  in  blood  plasma  as  an  ester 
of  oleic  and  palmitic  acids.  The  cholesterin 
of  nervous  tissues  is,  however,  free.  In 
bile  it  is  normally  present  in  small  quanti- 
ties only,  but  it  may  occur  in  excess,  and  so 
form  the  concretions  known  as  gallstones, 
which  are  generally  more  or  less  tinged 
with  bilirubin. 

Though  its  solubilities  remind  one  of  a 
fat,  cholesterin  is  not  a  fat,  but  a  monatomic 
alcohol,  probably  of  the  terpene  series.     Its  formula  is  C27H45.HO. 

From  alcohol  or  ether  containing  water  it  crystallises  in  the  form 
of  rhombic  tables,  which  contain  a  molecule  of  water  of  crystallisation  : 
these  are  easily  recognised  under  the  microscope  (fig.  25).  It  gives 
the  tests  described  under  the  practical  exercises  on  p.  79.  What  the 
physiological  uses  of  cholesterin  are  is  entirely  unknown. 

A  substance  called  iso-cholesterin,  isomeric  with  ordinary  chole- 
sterin, is  found  in  the  fatty  secretion  of  the  skin  (sebum)  :  it  is  largely 
contained  in  the  preparation  called  lanoline  made  from  sheep's-wool 
fat.     It  does  not  give  Salkowski's  reaction. 


Fl(i.  25.— Cholesterin  crystals. 


THE  USES   OF   BILE 

Bile  is  doubtless,  to  a  certain  extent,  excretory.  In  some  animals 
it  has  a  slight  action  on  fats  and  starch,  but  it  appears  to  be  rather  a 
coadjutor  to  the  pancreatic  juice  (especially  in  the  digestion  of  fat)  than 
to  have  any  independent  digestive  activity.  Its  auxiliary  action  in 
starch  digestion  has  been  shown  in  one  of  our  practical  exercises 
(p.  78).  It  has  a  similar  assisting  power  in  the  digestion  of 
proteins. 

Bile  is  said  to  be  a  natural  antiseptic,  lessening  the  putrefactive 
processes  in  the  intestine.  This  is  very  doubtful.  Though  the  bile 
salts  are  weak  antiseptics,  the  bile  itself  is  readily  putrescible,  and 
the  power  it  has  of  diminishing  putrescence  in  the  intestine  is  due 


94  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

chiefly  to  the  fact  that  by  increasing  absorption  it  lessens  the  amount 
of  putrescible  matter  in  the  bowel. 

When  the  bile  meets  the  chyme  the  turbidity  of  the  latter  is 
increased,  owing  to  the  precipitation  of  unpeptonised  protein.  This  is 
an  action  due  to  the  bile  salts,  and  it  has  been  surmised  that  this  con- 
version of  the  chyme  into  a  more  viscid  mass  is  to  hinder  somewhat 
its  progress  through  the  intestines  :  it  clings  to  the  intestinal  wall, 
thus  allowing  absorption  to  take  place.  The  neutralisation  of  the  acid 
gastric  juice  by  the  bile  also  allows  the  alkalinity  of  the  pancreatic 
juice  to  have  full  play.  Bile  is  a  solvent  of  fatty  acids,  and  assists 
the  absorption  of  fat. 

THE   FATE   OF  THE  BILIARY   CONSTITUENTS 

We  have  seen  that  fistula  bile  is  poor  in  solids  as  compared  with 
normal  bile,  and  that  this  is  explained  on  the  supposition  that  the 
normal  bile  circulation  is  not  occurring — the  liver  cannot  excrete 
what  it  does  not  receive  back  from  the  intestine.  Schiff  was  the  first 
to  show  that  if  the  bile  is  led  back  into  the  duodenum,  or  even  if  the 
animal  is  fed  on  bile,  the  percentage  of  solids  in  the  bile  excreted  is 
at  once  raised.  It  is  on  these  experiments  that  the  theory  of  a  bile 
circulation^ is  mainly  founded.  The  bile  circulation  relates,  however, 
chiefly,  if  not  entirely,  to  the  bile  salts  :  they  are  found  but  sparingly 
in  the  faeces  ;  they  are  only  represented  to  a  slight  extent  in  the  urine ; 
hence  it  is  calculated  that  seven-eighths  of  them  are  reabsorbed  from 
the  intestine.  Small  quantities  of  cholalic  acid,  taurine,  and  glycine 
are  found  in  the  faeces ;  the  greater  part  of  these  products  of  the 
decomposition  of  the  bile  salts  is  taken  by  the  portal  vein  to  the 
liver,  where  they  are  once  more  synthesised  into  the  bile  salts. 
Some  of  the  taurine  is  absorbed  and  excreted  as  tauro-carbamic 
acid  (C2H4NHCO.NH2HSO3)  in  the  urine.  Some  of  the  absorbed 
glycine  may  be  excreted  as  urea.  The  cholesterin  and  mucus  are 
found  in  the  faeces  ;  the  pigment  is  changed  into  stercobilin,  a  sub- 
stance like  hydrobilirubin.  Some  of  the  stercobilin  is  absorbed,  and 
leaves  the  body  as  the  urinary  pigment,  urobilin. 

THE   FiECES 

The  faeces  are  alkaline  in  reaction,  and  contain  the  following  sub- 
stances : — 

1.  Water :  in  health  from  68  to  82  per  cent. ;  in  diarrhoea  it  is 
more  abundant  still. 

2.  Undigested  food  :  that  is,  if  food  is  taken  in  excess,  some  escapes 


THE  DIGESTIVE  JUICES  96 

the  action  of  the  digestive  juices.     On  a  moderate  diet  unaltered 
protein  is  never  found. 

3.  Indigestible  constituents  of  the  food  :  cellulose,  keratin,  mucin, 
chlorophyll,  gums,  resins,  cholesterin. 

4.  Constituents  digestible  with  difficulty :  uncooked  starch,  ten- 
dons, elastin,  various  phosphates,  and  other  salts  of  the  alkaline  earths. 

5.  Products  of  decomposition  of  the  food  :  indole,  skatole,  phenol, 
acids  such  as  fatty  acids,  lactic  acid,  &c. ;  haematin  from  haemoglobin  ; 
insoluble  soaps  like  those  of  calcium  and  magnesium. 

6.  Bacteria  of  all  sorts  and  debris  from  the  intestinal  wall ;  cells, 
nuclei,  mucus,  &c.     This  forms  a  very  large  contribution. 

7.  Bile  residues  :  mucus,  cholesterin,  traces  of  bile  acids  and  their 
products  of  decomposition,  stercobilin  from  the  bile  pigment.^ 

MECONIUM 

Meconium  is  the  name  given  to  the  greenish-black  contents  of  the 
intestine  of  new-born  children.  It  is  chiefly  concentrated  bile,  with 
debris  from  the  intestinal  wall.  The  pigment  is  a  mixture  of  bilirubin 
and  biliverdin  ;  it  is  not  stercobilin. 

ABSORPTION 

Food  is  digested  in  order  that  it  may  be  absorbed.  It  is  absorbed 
in  order  that  it  may  be  assimilated — that  is,  become  an  integral  part 
of  the  living  material  of  the  body. 

Having  now  considered  the  action  of  digestive  juices,  we  can  study 
the  absorption  which  follows.  In  the  mouth  and  oesophagus  the 
thickness  of  the  epithelium  and  the  quick  passage  of  the  food  through 
these  parts  reduce  absorption  to  a  minimum.  Absorption  takes  place 
to  a  small  extent  in  the  stomach ;  the  small  intestine,  with  its  folds 
and  villi  to  increase  its  surface,  is,  however,  the  great  place  for  absorp- 
tion ;  and,  although  the  villi  are  absent  from  the  large  intestine, 
absorption  occurs  there  also,  but  to  a  less  extent. 

Foods  such  as  water  and  soluble  salts  like  sodium  chloride  are 
absorbed  unchanged.  The  organic  foods,  however,  are  considerably 
changed,  colloid  materials  like  starch  and  protein  being  converted  re- 
spectively into  the  diffusible  materials  sugar  and  amino-acids. 

There  are  two  channels  of  absorption,  the  blood  vessels  (portal 
capillaries)  and  the  lymphatic  vessels  or  lacteals. 

Absorption,  however,  is  no  mere  physical  process  of  diffusion  and 
filtration.     We  must  also  take  into  account  the  fact  that  the  cells 

'  Stercobilin  may  originate  also  from  the  heematin  of  the  food.     (MacMunn.) 


96  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

through  which  the  absorbed  subtances  pass  are  living,  and  in  virtue 
of  their  vital  activity  not  only  select  materials  for  absorption,  but  also 
change  those  substances  while  in  contact  with  them.  These  cells 
are  of  two  kinds :  (1)  the  columnar  epithelium  that  covers  the 
surface ;  and  (2)  the  lymph  cells  in  the  lymphoid  tissue  beneath.  It 
is  now  generally  accepted  that  of  the  two  the  former,  the  columnar 
epithelium,  is  the  more  important.  When  these  cells  are  removed,  or 
rendered  inactive  by  sodium  fluoride,  absorption  practically  ceases, 
though  the  opportunities  for  simple  filtration  or  diffusion  would  be  by 
such  means  increased. 

Absorption  of  Carbohydrates. — Though  the  sugar  formed  from 
starch  by  ptyalin  and  amylopsin  is  maltose,  that  found  in  the  blood 
is  glucose.  Under  normal  circumstances  little  if  any  is  absorbed  by 
the  lacteals.  The  glucose  is  formed  from  the  maltose  by  the  succus 
entericus,  and  perhaps  also  by  the  vital  action  of  the  epithelial  cells. 
Cane  sugar  and  milk  sugar  are  also  converted  into  glucose  before 
absorption. 

The  carbohydrate  food  which  enters  the  blood  as  glucose  is  taken 
to  the  liver,  and  there  stored  up  in  the  form  of  glycogen — a  reserve 
store  of  carbohydrate  material  for  the  future  needs  of  the  body. 
Glycogen,  however,  is  found  in  animals  which  take  no  carbohydrate 
food.  It  must  then  be  formed  by  the  protoplasmic  activity  of  the 
liver  cells  from  their  protein  constituents.  The  carbohydrate  store 
leaves  the  liver  in  the  blood  of  the  hepatic  vein  as  glucose  (dextrose) 
once  more. 

The  above  is  a  brief  statement  of  the  glycogenic  functions  of  the 
liver  as  taught  by  Claude  Bernard,  and  accepted  by  the  majority  of 
physiologists.  It  has  always  been  strongly  contested  by  Pavy,  who 
holds  that  the  glycogen  formed  in  the  liver  from  the  sugar  of  the 
portal  blood  is  never  during  life  reconverted  into  sugar,  but  is  used 
in  the  formation  of  other  substances  like  fat  and  protein ;  in  support 
of  this  he  has  shown  that  proteins  contain  a  carbohydrate  radical. 
He  denies  that  the  post-mortem  formation  of  sugar  from  glycogen 
that  occurs  in  an  excised  liver  is  a  true  picture  of  what  occurs  during 
hfe. 

Absorption  of  Proteins. — It  is  possible  that  under  abnormal  con- 
ditions a  certain  amount  of  soluble  protein  is  absorbed  unchanged. 
Thus,  after  eating  a  large  number  of  eggs,  egg-albumin  has  been 
stated  to  be  discoverable  in  the  urine.  Patients  fed  per  rectum  derive 
nourishment  from  protein  food,  although  proteolytic  ferments  are  not 
present  in  this  part  of  the  intestine. 

Under  normal  conditions,  however,  the  food-proteins  are  broken 


THE  DIGESTIVE  JIHCES  97 

up  into  substances  with  smaller  molecules,  and  the  ready  diffusibility 
of  peptones  led  most  physiologists  to  consider  that  protein  was  usually 
absorbed  as  peptone,  or  as  proteose  and  peptone.  But  proteose 
and  peptone  are  absent  from  the  blood  and  lymph  under  all  circum- 
stances, even  from  the  portal  blood  during  the  most  active  digestion. 
It  is  fortunate  that  this  is  so,  for  proteose  and  peptone  when  intro- 
duced into  the  blood  produce  poisonous  effects  ;  the  coagulability  of 
the  blood  is  lessened,  blood  pressure  falls,  secretion  ceases,  and  in 
the  dog  0"3  gramme  of  commercial  peptone  per  kilogramme  of  body- 
weight  is  often  sufficient  to  produce  death. 

This  absence  of  '  peptone '  (using  the  word  to  include  the  prote- 
oses) did  not,  however,  absolutely  negative  the  idea  that  '  peptone ' 
is  the  form  in  which  proteins  are  absorbed,  and  the  difficulty  was  met 
by  supposing  that  during  absorption  the  products  of  proteolysis  were 
reconverted  into  native  proteins  (albumins  and  globulins).  This 
synthesis  was  further  considered  to  be  accomplished  by  the  epithelial 
cells  that  line  the  intestine. 

This  view  has  now  had  its  day,  and  the  change  of  opinion  that 
has  relegated  it  to  the  past  is  due  (1)  to  our  increased  knowledge  of 
the  power  of  trypsin  and  erepsin  ;  (2)  to  a  more  careful  examination 
of  the  intestinal  contents,  and  of  the  blood  during  absorption.  We 
now  know  that  in  the  intestine  the  proteins  are,  by  the  two  enzymes 
trypsin  and  erepsin,  broken  down  beyond  the  peptone  stage  into 
their  final  cleavage  products,  the  amino-acids,  and  that  these  pro- 
bably pass  into  the  blood  as  such,  for  the  amount  of  non-protein 
nitrogen  in  that  fluid  is  increased  during  absorption.  These  amino- 
acids  are  partly  utilised  by  the  cells  of  the  body  to  repair  their 
waste,  but  partly  and  to  a  still  greater  extent  converted  by  the  liver 
into  the  waste  substance  urea,  which  is  finally  excreted  by  the 
kidneys.  The  view  that  the  absorptive  epithelium  of  the  alimentary 
tract  has  any  special  power  in  building  up  proteins  from  these  simple 
cleavage  products  has  not  been  confirmed.  If  an  animal  is  fed  on 
the  cleavage  products  obtained  from  a  pancreatic  digest  nitrogenous 
equilibrium  is  still  maintained. 

We  thus  see  that  the  cells  of  the  body  possess  the  power  of 
rebuilding  the  proteins  peculiar  to  themselves  from  the  fragments 
of  the  molecules  of  the  food  proteins.  This  accounts  for  the  fact  that 
the  animal  tissues  retain  their  chemical  individuality  in  spite  of  the 
great  variations  in  the  composition  of  the  diet  the  animal  takes. 

If  a  man  wishes  to  build  a  new  house,  and  to  employ  for  the 
purpose  the  bricks  previously  used  in  the  building  of  another  house, 
he  takes  the  old  house  to  pieces  and  uses  the  bricks  and  stones  most 

H 


98  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

appropriate  for  his  purpose,  rearranges  them  in  such  a  way  that  the 
new  house  has  its  own  special  architectural  features,  and  discards  as 
waste  the  bricks  and  stones  which  are  not  suitable.  This  idea  under- 
lies the  term  so  often  used  by  German  writers,  who  speak  of  the 
cleavage  products  of  protein  as  Bausteine  (building  stones).  Each 
tissue  has  special  architectural  features  in  its  protein  molecules,  and 
these  molecules  are  reconstructed  by  using  the  Bausteine  that  pre- 
viously had  been  used  in  the  building  of  other  protein  molecules, 
either  in  another  animal  or  in  vegetable  structures.  The  Bausteine 
which  are  in  excess  or  are  unsuitable  are  simply  got  rid  of  as  waste 
substance. 

A  large  number  of  the  Bausteine  are  never  actually  built  into 
protoplasm,  but  are  converted  by  the  liver  into  urea,  and  this  is  dis- 
charged from  the  body  via  the  urine  (see  Urea  formation,  p.  148). 


Fig.  26.— Section  of  the  villus  of  a  rat  killed  during  fat-absorption  (PI  A.  Schafer)  : 
ep,  epithelium  ;  str,  striated  border ;  c,  lympb  cells ;  c',  lymph  cells  in  the  epithelium  ; 
Z,  central  lacteal  containing  disintegrating  lymph  cells. 

One  can  only  conjecture  at  present  which  are  the  ones  that  on 
p.  52  we  compared  to  diamonds,  because  they  are  unusually  precious 
for  the  synthesis  of  protein  by  tissue  cells  ;  but  probably  phenyl- 
alanine and  its  near  relation  tyrosine  come  into  this  category,  for  if 
they  are  injected  into  the  blood-stream  they  do  not  give  rise  to  any 
increase  in  the  urea  formed. 


THE  DIGESTIVE  JUICES 


99 


Absorption  of  fats. — The  fats  undergo  in  the  intestine  two 
changes  :  one  a  physical  change  (emulsification),  the  other  a  chemical 
change  (saponification).  The  lymphatic  vessels  are  the  great  channels 
for  fat-absorption,  and  their  name,  lacteals,  is  derived  from  the 
milk-like  appearance  of  their  contents  (chyle)  during  the  absorption 
of  fat. 

The  way  in  which  the  minute  fat  globules  pass  from  the  intestine 
into  the  lacteals  has  been  studied  by  killing  animals  at  varying  periods 
after  a  meal  of  fat  and  making  osmic  acid  microscopic  preparations 
of  the  villi.     Figs.  26  and  27  illustrate  the  appearances  observed. 

The  columnar  epithelium  cells  become  first  filled  with  fatty 
globules  of  varying  size,  which  are  generally  larger  near  the  free 
border.  The  globules  pass  down  the  cells,  the  larger  ones  breaking 
up  into  smaller  ones  during  the  journey  ;  they  are  then  transferred 
to  the  amoeboid  cells  of  the  lymphoid 
tissue  beneath  :  these  ultimately  pene- 
trate into  the  central  lacteal,  where 
they  either  disintegrate  or  discharge 
their  cargo  into  the  lymph  stream. 
The  globules  are  by  this  time  divided 
into  immeasurably  small  ones,  the 
molecular  basis  of  chyle.  The  chyle 
enters  the  blood  stream  by  the  thoracic 
duct,  and  after  an  abundant  fatty  meal 

,11111  •  •,  -ii  ,1       Fm.27. — Mucous  membrane  of  frog's  intestiue 

the   blood    plasma  is  quite  milky  ;   the        during  fat-absorption  (E.  a.  Scliafer) :  ep, 

fat  droplets  are  so  small  that  they  t^^^^A:!'"'''^'^''''''''''  '''''^^'' 
circulate  without  hindrance  through 

the  capillaries.  The  fat  in  the  blood  after  a  meal  is  eventually  stored 
up  in  connective  tissue  cells  of  adipose  tissue.  It  must,  however, 
be  borne  in  mind  that  the  fat  of  the  body  is  not  exclusively  derived 
from  the  fat  of  the  food,  but  it  may  originate  also  from  carbo- 
hydrates, and  possibly,  in  the  opinion  of  some  physiologists,  from 
protein  as  well. 

As  the  fat  globules  were  never  seen  penetrating  the  striated 
border  of  the  epithelial  cells,  there  was  a  difficulty  in  understanding 
how  they  reached  the  interior  of  these  cells ;  the  cells  will  not  take 
up  other  particles,  and  it  is  certain  that  they  do  not  in  the  higher 
animals  protrude  pseudopodia  from  their  borders  (this,  however,  does 
occur  in  the  endoderm  of  some  of  the  lower  invertebrates) ._ 

Eecent  research  has  solved  this  difficulty;,  ,Li  ilhe  ^rst-.  place 
particles  may  be  present  in  the  epithelium"  arnd  lymphoid  cells  while 
no  fat  is  being  absorbed.     These  parti<?]es;are'5roi6pla2imic  in  nature, 


100  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

as  they  stain  with  reagents  that  stain  protoplasmic  granules ;  but  as 
they  also  stain  darkly  with  osmic  acid,  they  are  apt  to  be  mistaken 
for  fat.  There  is,  however,  no  doubt  that  the  particles  found  during 
fat-absorption  are  composed  of  fat.  There  is  also  no  doubt  that  the 
epithelial  cells  have  the  power  of  again  forming  fat  out  of  the  fatty 
acids  and  glycerin  into  which  it  has  been  broken  up  in  the  intestine. 
Munk,  who  performed  a  large  number  of  experiments  on  the  subject, 
showed  that  the  splitting  of  fats  into  glyceiin  and  fatty  acids  occurs 
to  a  much  greater  extent  than  was  formerly  supposed  :  these  sub- 
stances, being  soluble,  pass  readily  into  the  epithelium  cells,  and  these 
cells  perform  the  synthetic  act  of  building  them  into  fat  once  more ; 
the  fat  so  formed  appears  in  the  form  of  small  globules,  surrounding 
or  becoming  mixed  with  the  protoplasmic  granules  that  are  ordinarily 
present.  Another  remarkable  fact  which  he  made  out  is  that  after 
feeding  an  animal  on  fatty  acids  the  chyle  contains  fat.  The 
necessary  glycerin  must  have  been  formed  by  protoplasmic  activity 
during  absorption.  The  more  recent  work  of  Moore  and  Eockwood 
has  shown  that  fat  is  absorbed  entirely  as  glycerin  and  either  fatty 
acid  or  soap ;  and  that  prehminary  emulsification,  though  advan- 
tageous for  the  formation  of  these  substances,  is  not  essential. 

Bile  aids  the  digestion  of  fat,  in  virtue  of  its  being  a  solvent  of  fatty 
acids,  and  it  probably  assists  fat-absorption  by  reducing  the  surface 
tension  of  the  intestinal  contents  ;  membranes  moistened  with  bile 
allow  fatty  materials  to  pass  through  them  more  readily  than  would 
otherwise  be  the  case.  In  cases  of  disease  in  which  bile  is  absent 
from  the  intestines  a  large  proportion  of  the  fat  in  the  food  passes 
into  the  faeces. 


LESSON   IX 
THE  BLOOD  AND  RESPIRATION 

[Blood  Plasma. 

1.  The  coagulation  of  the  blood  has  been  prevented  in  specimen  A  by  the 
addition  of  neutral  salt  (an  equal  volume  of  saturated  sodium- sulphate 
solution,  or  a  quarter  of  its  volume  of  saturated  magnesium-sulphate  solu- 
tion). The  corpuscles  have  settled,  and  the  supernatant  salted  plasma  has 
been  siphoned  off. 

2.  The  coagulation  of  the  blood  in  specimen  B  has  been  prevented  by  the 
addition  of  an  equal  volume  of  ai  0'4-per-cent.  solution  of  potassium  oxalate 
in  normal  saline  solution. 

3.  Put  a  small  quantity  of  A  into  three  test-tubes  and  dilute  each  with 
about  ten  times  its  volume  of  liquid  : 

A  1.  With  distilled  water. 

A  2.  With  solution  of  fibrin  ferment  containing  a  little  calcium  chloride.^ 

A  3.  With  the  same. 

4.  Put  A  1  and  A  2  into  the  water-bath  at  40°  C. ;  leave  A  3  at  the  tem- 
perature of  the  air.  A  1  coagulates  slowly  or  not  at  all ;  A  2  coagulates 
rapidly :  A  3  coagulates  less  rapidly  than  A  2. 

5.  Add  to  some  of  B  a  few  drops  of  dilute  (2  per  cent.)  calcium-chloride 
solution  :  it  coagulates,  and  more  quickly,  if  the  temperature  is  40°  C. 

Blood  Serum. 

Blood  serum  is  the  lluid  residue  of  the  blood  after  the  separation  of  the 
clot ;  it  is  blood  plasma  minus  the  fibrin  which  it  yields.  The  general  ap- 
pearance of  fibrin  obtained  by  whipping  fresh  blood  will  already  be  familiar 
to  the  student,  as  he  has  used  it  in  experiments  on  digestion. 

Serum  has  a  yellowish  tinge  due  to  serum  lutein,  but  as  generally  obtained 
it  is  often  contaminated  with  a  small  amount  of  oxyhaemoglobin,  and  so  looks 
reddish.  It  contains  proteins  (giving  the  general  tests  already  studied  in 
Lesson  IV.),  extractives,  and  salts  in  solution.  The  proteins  are  serum 
albumin  and  serum  globulin.  The  fibrin  ferment  is  also  a  protein-like  sub- 
stance. It  is  present  in  only  small  quantities,  and  in  the  following  experi- 
ments is  precipitated  with  serum  globulin. 

Separation  of  the  serum  proteins,  —{a)  Dilute  serum  with  fifteen  times 
its  volume  of  water.     It  becomes  cloudy  owing  to  the  partial  precipitation  of 

'  An  easy  way  of  preparing  an  impure  but  efficient  solution  of  fibrin  ferment 
is  to  take  5  c.c.  of  blood  serum  and  dilute  it  with  a  litre  of  distilled  water.  A 
partial  precipitation  of  globulin  takes  place,  and  carries  down  the  ferment  with  it. 
After  a  few  hours  pour  off  the  supernatant  fiuid  and  dissolve  the  precipitate  in 
half  a  litre  of  tap-water  to  which  a  few  drops  of  2-per-cent.  solution  of  calcium 
chloride  have  been  added.  The  solution  can  be  then  given  round  to  the  class  as 
fibrin  ferment. 


102  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

serum  globulin.     Add  a  few  drops  of  2-per-cent.  acetic  acid ;  the  precipitate 
becomes  more  abundant,  but  dissolves  in  excess  of  the  acid. 

(6)  Pass  a  stream  of  carbonic  acid  through  serum  diluted  with  twenty 
times  its  bulk  of  water.     A  partial  precipitation  of  serum  globulin  occurs. 

(c)  Saturate  some  serum  with  magnesium  sulphate  by  adding  crystals  of 
the  salt  and  grinding  in  a  mortar.  A  precipitate  of  serum  globulin  is  pro- 
duced. 

(d)  Half  saturate  the  serum  with  ammonium  sulphate  by  adding  to  it  an 
equal  volume  of  a  saturated  solution  of  the  salt.  Serum  globulin  is  pre- 
cipitated. 

(e)  Completely  saturate  the  serum  with  ammonium  sulphate  by  adding 
crystals  of  the  salt  and  grinding  in  a  mortar ;  a  precipitate  is  produced  of 
both  the  globulin  and  the  albumin.  Filter  through  a  dry  filter  paper ;  the 
filtrate  contains  no  protein. 

Haemoglobin. 

6.  Direct  the  spectroscope  to  the  window  and  carefully  focus  Fraunhofer's 
lines.  Note  especially  D  in  the  yellow,  and  E,  the  next  well-marked  line,  in 
the  green. 

7.  Direct  the  spectroscope  to  a  luminous  gas  flame  :  these  lines  are 
absent.  Place  a  little  sodium  chloride  in  the  flame.  Notice  the  bright  yellow 
line  in  the  position  of  the  D  line. 

8.  Take  a  series  of  six  test-tubes  of  about  equal  size.  Fill  the  first  with 
diluted  defibrinated  ox- blood  (1  part  of  blood  to  81  of  water)  ;  then  fill  the 
second  tube  with  the  same  mixture  diluted  with  an  equal  bulk  of  water 
(1  in  64)  ;  half  fill  the  third  tube  with  this  and  fill  up  the  tube  with  an  equal 
bulk  of  water  (1  in  128),  and  so  on.  The  sixth  tube  will  contain  1  part  of 
blood  to  1,024  of  water,  and  will  be  nearly  colourless. 

9.  Into  another  series  of  six  test-tubes  put  a  few  drops  of  ammonium 
sulphide ;  then  pour  in  some  of  the  contents  of  each  of  the  first  series  and 
warm  very  gently. 

10.  Examine  the  tubes  with  the  spectroscope  and  map  out  on  a  chart  the 
typical  absorption  bands  of  oxyhaemoglobin  in  the  first  series,  and  of  (reduced) 
haemoglobin  in  the  second  series.  Notice  that  in  the  more  dilute  specimens 
of  haemoglobin  the  bands  are  no  longer  seen,  whereas  those  of  oxyhaemoglobin 
in  specimens  similarly  diluted  are  still  visible. 

11.  Take  a  tube  which  shows  the  single  band  of  reduced  haemoglobin 
and  shake  it  with  the  air ;  the  bright  red  colour  returns  to  it  and  it  shows 
spectroscopically  the  two  bands  of  oxyhaemoglobin  for  a  short  time.  Con- 
tinue watching  the  two  bands,  and  note  that  they  fade  and  are  replaced  by 
a  single  band  as  reduction  again  occurs. 

12.  Mix  a  drop  of  defibrinated  rat's  blood  on  a  slide  witli  a  drop  of  water, 
or  mount  it  in  a  drop  of  Canada  balsam.  Examine  the  crystals  of  oxyhaemo- 
globin as  they  form. 

13.  Smear  a  little  blood,  obtained  by  pricking  the  finger,  on  a  slide,  and 
allow  it  to  dry  ;  cover,  and  run  glacial  acetic  acid  under  the  cover  glass,  and 
boil.     Examine  microscopically  for  the  dark  brown  crystals  of  haemin. 


COAGULATION   OF  BLOOD 

Microscopic  investigation  of  vertebrate  blood  shows  that  it  consists 
of  a  fluid  which  holds  in  suspension  large  numbers  of  solid  bodies — 
the  red  and  the  white  corpuscles  and  the  blood  platelets. 

After  blood  is  shed  it  rapidly  becomes  viscous  and  then  sets  into 


THE   BLOOD  103 

a  firm  red  jelly.  The  jelly  soon  contracts  and  squeezes  out  a  straw- 
coloured  fluid  called  the  serum,  in  which  the  shrunken  clot  ultimately 
floats. 

With  the  microscope,  filaments  of  fibrin  are  seen  forming  a  net- 
work throughout  the  fluid,  many  radiating  from  small  clumps  of 
blood  platelets.  It  is  the  formation  of  fibrin  which  is  the  essential 
act  of  coagulation  :  this  entangles  the  corpuscles  and  forms  the  clot. 
Fibrin  is  formed  from  the  plasma,  and  may  be  obtained  free  from 
corpuscles  when  blood  plasma  is  allowed  to  clot,  the  corpuscles  having 
previously  been  removed.  It  may  also  be  obtained  from  blood  by 
whipping  it  with  a  bunch  of  twigs ;  the  fibrin  adheres  to  the  twigs 
and   entangles   but   few   corpuscles.      These    may   be    removed    by 


Fig.  28.— Fibrin  filaments  and  blood  platelets  :  A,  network  of  fibrin  sbown  after  washing  away  the 
corpuscles  from  a  preparation  of  blood  that  has  been  allowed  to  clot.  Many  of  the  filaments 
radiate  from  small  clumps  of  blood  platelets.  B  (from  Osier),  blood  corpuscles  and  blood  platelets 
within  a  small  vein. 

washing  with  water.  Serum  is  plasma  minus  the  fibrin  which  it 
yields.  The  relation  of  plasma,  serum,  and  clot  can  be  seen  at  a 
glance  in  the  following  scheme  of  the  constituents  of  the  blood  :— 


Blood 


Plasma  11^^^.^, 
Corpuscles         ) 


It  may  be  roughly  stated  that  in  100  parts  by  weight  of  blood  60-65 
parts  consist  of  plasma  and  35-40  of  corpuscles. 

The  buffy  coat  is  seen  when  blood  coagulates  slowly,  as  in  horse's 
blood.  The  red  corpuscles  sink  more  rapidly  than  the  white,  and 
the  upper  stratum  of  the  clot  (buffy  coat)  consists  mainly  of  fibrin 
and  white  corpuscles. 

Coagulation  is  hastened  by — 

1.  A  temperature  a  little  over  that  of  the  body. 

2.  Contact  with  foreign  matter. 

3.  Injury  to  the  vessel  walls. 

4.  Agitation. 

5.  Addition  of  calcium  salts. 

6.  Injection  of   nucleo-protein   produces    intravascular    clotting 


104  ESSENTIALS  OF  CHEMICAL  PHYSIOLOaY 

(positive  phase).     Very  minute  doses,  however,  produce  the  opposite 
effect :  namely,  delay  of  coagulation  (negative  effect). 
Coagulation  is  hindered  or  prevented  by — 

1.  A  low  temperature.  In  a  vessel  cooled  by  ice,  coagulation  may 
be  prevented  for  an  hour  or  more. 

2.  The  addition  of  a  large  quantity  of  neutral  salts,  like  sodium 
sulphate  or  magnesium  sulphate. 

3.  Addition  of  a  soluble  oxalate,  fluoride  or  citrate. 

4.  Injection  of  commercial  peptone  (which  consists  chiefly  of 
proteoses)  into  the  circulation  of  the  living  animal. 

5.  Addition  of  leech  extract  to  the  blood,  or  injection  of  leech 
extract  into  the  circulation  while  the  animal  is  alive. 

6.  Contact  with  the  living  vascular  walls. 

7.  Contact  with  oil. 

The  cause  of  the  coagulation  of  the  blood  may  be  briefly  stated  as 
follows  : — 

When  blood  is  within  the  vessels  one  of  the  constituents  of  the 
plasma,  a  protein  of  the  globulin  class  called  fibrinogen,  exists  in  a 
soluble  form. 

When  the  blood  is  shed  the  fibrinogen  molecule  is  altered  in  such 
a  way  that  it  gives  rise  to  the  comparatively  insoluble  material yi6ri?t. 
The  statement  has  been  made  that  the  fibrinogen  molecule  is  split 
into  two  parts  :  one  part  is  a  globulin  (fibrinoglobulin),  which  remains 
in  solution ;  the  other  and  larger  part  is  the  insoluble  substance 
fibrin.  It  is,  however,  doubtful  if  this  really  represents  what 
occurs,  for  recent  work  seems  to  show  that  the  fibrinoglobulin  is 
not  a  product  of  fibrinogen,  but  exists  in  the  blood  plasma  before- 
hand. At  any  rate,  whether  this  is  so  or  not,  the  fact  remains 
that  fibrin  is  the  important  product  and  the  only  one  which  need 
concern  us. 

The  next  question  is.  What  causes  the  transformation  of  fibrinogen 
into  fibrin  ?  And  the  answer  to  that  is,  that  the  change  is  due  to  the 
activity  of  a  special  unorganised  ferment  which  is  called yi^rm/crmew^ 
or  tkromhin. 

This  ferment  does  not  exist  in  healthy  blood  contained  in  healthy 
blood  vessels,  but  is  formed  by  the  disintegration  of  the  blood  platelets 
and  colourless  corpuscles  which  occurs  when  the  blood  leaves  the 
blood  vessels  or  comes  into  contact  with  foreign  matter.  Hence  the 
blood  does  not  coagulate  during  life.  But,  it  will  be  said,  disintegra- 
tion of  the  blood  corpuscles  occurs  during  life ;  why,  then,  does  the 
blood  not  coagulate  ?  The  reason  is  that  although  the  formed  elements 
do  disintegrate  in  the  living  blood,  such  a  phenomenon  takes  place 


THE  BLOOD  105 

very  slowly  and  gradually,  so  that  there  can  never  in  normal  cir- 
cumstances be  any  massive  liberation  of  fibrin  ferment,  and,  further, 
that  there  are  agencies  at  work  to  neutralise  the  fibrin  ferment  as  it  is 
formed.  The  most  noteworthy  of  these  neutralising  agencies  is 
the  presence  in  the  blood  of  an  antiferment  called  antithrombin, 
analogous  to  the  antipepsin  and  antitrypsin  which,  we  have  seen,  are 
efiQcacious  in  preventing  the  stomach  and  intestines  from  undergoing 
self-digestion. 

Thrombin  or  fibrin  ferment  belongs  to  the  class  of  nucleo-proteins, 
and  other  nucleo-proteins  (see  pp.  45,  46)  obtained  from  most  of  the 
cellular  organs  of  the  body  produce  intravascular  clotting  when  in- 
jected into  the  circulation  of  a  living  animal.  In  certain  diseased 
conditions  intravascular  clotting,  or  thrombosis,  sometimes  occurs. 
This  must  be  due  either  to  the  entrance  of  nucleo-protein  into  the 
circulation  from  diseased  tissues,  or  to  a  failure  of  the  body  to  pro- 
duce sufficient  antithrombin  to  neutralise  its  effect,  or  to  both  of 
these  conditions  together. 

Thrombin  is  believed  to  originate  chiefly  from  the  blood  platelets 
and  in  part  from  the  leucocytes.  Birds'  blood  clots  very  slowly,  and 
the  absence  of  blood  platelets  in  this  variety  of  blood  will  in  part 
account  for  this.  Lymph  which  contains  colourless  corpuscles,  but 
no  platelets,  also  clots  in  time,  so  in  this  case  the  colourless  corpuscles 
must  be  the  source  of  the  ferment.  One  should,  however,  be  careful 
in  speaking  of  the  disintegration  of  leucocytes  to  remember  that  the 
word  disintegration  does  not  mean  complete  breakdown,  leading  to 
disappearance  ;  the  colourless  corpuscles  do  not  appreciably  diminish 
in  number  when  the  blood  is  shed,  but  what  occurs  in  the  surviving 
leucocytes  is  a  shedding  out  of  certain  products,  among  which  fibrin- 
ferment  is  one. 

We  have  now  traced  fibrin-formation,  the  essential  cause  of  blood- 
clotting,  to  the  activity  of  thrombin ;  it  is  next  necessary  to  allude 
to  what  has  been  discovered  in  relation  to  the  origin  of  thrombin. 
Like  other  ferments,  it  is  preceded  by  a  mother  substance  or 
zymogen.  This  zymogen  is  called  prothrombin,  or  thrombogen,  and 
there  appear  to  be  two  necessary  agents  concerned  in  the  con- 
version of  thrombogen  into  thrombin  :  one  of  these  is  the  action 
of  calcium  salts,  the  other  is  the  presence  of  an  activating  agent, 
analogous  to  entero-kinase  (see  p.  84),  and  called  thrombo-hinase. 
The  exact  role  played  by  each  is  still  a  matter  of  speculation,  but 
we  may  learn  a  good  deal  by  studying  a  little  more  in  detail 
some  of  the  methods  already  enumerated  for  preventing  the  blood 
from  coagulating. 


106  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

The  part  played  by  calcium  salts  is  well  illustrated  by  the  fact 
that  coagulation  is  prevented  by  the  decalcification  of  the  blood. 
This  can  be  accomplished  by  the  addition  of  a  small  amount  of 
soluble  oxalate  or  fluoride  to  the  blood  immediately  it  is  shed.  The 
calcium  of  the  blood  plasma  is  then  immediately  precipitated  as 
insoluble  calcium  oxalate  or  fluoride,  and  is  thus  not  available  for 
the  transformation  of  thrombogen  into  thrombin.  The  addition  of 
the  oxalate  or  fluoride  must  be  rapidly  performed,  otherwise  time  will 
be  given  for  the  conversion  of  thrombogen  into  thrombin,  and 
thrombin,  when  formed,  will  act  upon  fibrinogen  whether  the  calcium 
has  been  removed  or  not.  In  other  words,  calcium  is  only  necessary 
for  the  formation  of  fibrin-ferment,  and  not  for  the  action  of  fibrin- 
ferment  on  fibrinogen.  Fibrin  is  thus  not  a  compound  of  calcium 
and  fibrinogen. 

The  action  of  a  soluble  citrate  is  also  in  a  certain  sense  a 
decalcifying  action,  for  although  calcium  citrate  is  a  soluble  salt  it 
does  not  ionise  in  solution  so  as  to  liberate  the  free  calcium  ions 
which  are  essential  for  thrombin  formation. 

Oxalated  blood  (or  oxalated  plasma)  will  clot  when  the  calcium  is 
once  more  restored  by  the  addition  of  a  small  amount  of  calcium 
chloride,  but  such  an  addition  to  fluoride  plasma  will  not  induce  clot- 
ting ;  thrombin  must  be  added  also.  In  some  way  sodium  fluoride 
interferes  with  the  formation  of  thrombin,  probably  by  preventing  the 
liberation  of  thrombo-kinase  from  the  formed  elements  of  the  blood. 

The  other  activating  agent,  thrombo-kinase,  is  in  part  liberated 
from  the  formed  elements  of  the  blood,  but  it  is  also  obtained  from 
many  other  tissues.  If  a  haemorrhage  takes  place  under  ordinary 
circumstances  the  blood  as  it  flows  from  the  wound  passes  over  the 
muscles  and  skin  that  have  been  cut,  and  rapidly  clots  owing  to  the 
thrombo-kinase  supplied  by  those  tissues.  If  blood  is  obtained  by 
drawing  it  off  through  a  perfectly  clean  cannula  into  a  clean  vessel 
without  allowing  it  to  touch  the  tissues,  it  remains  unclotted  for  a 
long  time  ;  in  the  case  of  birds'  blood  this  time  may  extend  to  many 
days ;  but  the  addition  of  a  small  piece  of  tissue  such  as  muscle,  or  of 
an  extract  of  such  a  tissue,  produces  almost  immediate  clotting.  If  a 
solution  of  fibrinogen  is  prepared  and  calcium  added  it  will  not  clot ; 
if  thrombin  or  a  fluid  such  as  serum  which  contains  thrombin  is  added 
also  it  will  clot.  It  will  not  clot  if  birds'  plasma  obtained  as  above  is 
added  to  it ;  nor  if  tissue  extract  is  added  to  it ;  but  if  both  are  added 
it  will.  In  other  words,  the  thrombogen  of  the  birds'  plasma 
plus  the  thrombo-kinase  of  the  tissue  extract  have  the  same  effect  as 
thrombin. 


THE   BLOOD  107 

The  next  point  to  consider  is  why  blood  obtained  after  the  previous 
injection  of  proteoses  (or  commercial  peptone)  into  the  circulation 
should  not  clot.  It  certainly  contains  calcium  salts,  and  probably 
both  thrombogen  and  thrombo-kinase,  for  it  can  be  made  to  clot  with- 
out the  addition  of  either  ;  for  instance,  by  dilution  or  the  passage  of 
a  stream  of  carbon  dioxide  through  it.  There  must  be  something  in 
peptone  blood  which  antagonises  the  action  of  thrombin.  This  some- 
thing is  an  excess  of  antithrombin.  Peptone  will  not  hinder  blood 
coagulation,  or  only  very  slightly  if  it  is  added  to  the  blood  after  it  is 
shed.  The  antithrombin  must  therefore  have  been  added  to  the 
blood  while  it  was  circulating  in  the  body.  We  can  even  go  further 
than  this  and  say  what  part  of  the  body  it  is  which  is  concerned  in  the 
production  of  antithrombin :  it  is  the  liver,  for  if  the  liver  is  shut 
off  from  the  circulation,  peptone  is  ineffective  in  its  action.  The 
converse  experiment  confirms  this  conclusion,  for  if  a  solution  of 
peptone  is  artificially  perfused  through  an  excised  surviving  liver,  a 
substance  is  formed  which  has  the  power  of  hindering  or  preventing 
the  coagulation  of  shed  blood. 

We  are  thus  justified  in  two  conclusions  : — 

(1)  That  the  antithrombin  which  is  normally  present  in  healthy 
blood  in  sufficient  quantities  to  prevent  intravascular  clotting,  is 
formed  in  the  liver. 

(2)  That  commercial  peptone  in  virtue  of  the  proteoses  it  contains 
stimulates  this  action  of  the  liver  to  such  an  extraordinary  degree, 
that  the  accumulation  of  antithrombin  in  the  blood  becomes  suffi- 
ciently great  to  prevent  the  blood  from  clotting  even  after  it  is  shed. 

It  should  be  noted,  however,  that  this  effect  upon  the  Uver  varies 
in  different  animals,  and  is  most  marked  in  the  dog. 

We  shall  conclude  by  considering  only  one  more  of  the  hindrances 
to  coagulation,  and  that  by  no  means  the  least  interesting.  The 
leech  lives  by  sucking  the  blood  of  other  animals ;  from  the  leech's 
point  of  view^  it  is  therefore  necessary  that  the  blood  should  flow 
freely  and  not  clot.  The  glands  at  the  head  end  of  the  leech,  often 
spoken  of  roughly  as  its  salivary  glands,  secrete  something  which 
hinders  the  blood  from  coagulating,  and  everyone  knows  by  experience 
who  has  been  treated  by  leeches  how  difficult  it  is  to  prevent  a  leech- 
bite  from  bleeding  after  the  leech  has  been  removed ;  complete 
cleansing  is  necessary  to  wash  away  the  leech's  secretion  from  the 
wound.  Now  if  an  extract  of  leeches'  heads  is  made  with  salt  solution 
and  filtered,  that  fluid  will  prevent  coagulation,  whether  it  is  injected 
into  the  blood-stream  or  added  to  shed  blood.  The  substance  in  the 
extract  is  called  hirudin,  and  this  is  believed  to  be  antithrombin  itself. 


108 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


We    may   summarise   our  present    knowledge   of  the   causes   of 
coagulation  in  the  following  tabular  way : — 


From  the  platelets, 
and  to  a  lesser  degree 
from  the  leucocytes,  a 
nucleo-protein  is  shed 
out  called 


Thrombogen. 


From  the  formed 
elements  of  the  blood, 
but  also  from  the  tissues 
over  which  the  escaping 
blood  Hows,  is  shed  out 
an  activating  agent 
called 
Thrombo-kinase. 


In  the  blood  plasma 
a  protein  substance  ex- 
ists caUed 

Fibrinogen. 


In  the   presence  of  calcium  salts,  thrombo- 
kinase  activates  thrombogen  in  such  a  way  that 
an  active  ferment  is  produced,  which  is  called 
Thrombin. 


Thrombin  or  fibrin -ferment  acts  on  fibrinogen  in  such  a  way  that  it  is 
transformed  into  the  insoluble  stringy  material  which  is  called 
Fibrin. 


THE  PLASMA  AND   SERUM 

The  liquid  in  which  the  corpuscles  float  may  be  obtained  by 
employing  one  or  other  of  the  methods  already  described  for  pre- 
venting the  blood  from  coagulating.  The  corpuscles,  being  heavy, 
sink,  and  the  supernatant  plasma  can  then  be  removed  by  a  pipette 
or  siphon  ;  the  separation  can  be  effected  more  thoroughly  by  the 
use  of  a  centrifugal  machine  (see  fig.  60,  Lesson  XXI.). 

On  counteracting  the  influence  which  has  prevented  the  blood  from 
coagulating,  the  plasma  then  itself  coagulates.  Thus  plasma  obtained 
by  the  use  of  cold,  clots  on  warming  gently ;  plasma  which  has  been 
decalcified  by  the  action  of  a  soluble  oxalate  clots  on  the  addition  of 
a  calcium  salt ;  plasma  obtained  by  the  use  of  a  strong  solutfon  of 
salt  coagulates  when  this  is  diluted  by  the  addition  of  water,  the 
addition  of  fibrin  ferment  being  necessary  in  most  cases ;  where 
coagulation  occurs  without  the  addition  of  fibrin-ferment,  no  doubt 
some  is  present  from  the  partial  disintegration  of  the  corpuscles  which 
has  already  occurred.  Pericardial  and  hydrocele  fluids  resemble  pure 
plasma  very  closely  in  composition.  As  a  rule,  however,  they  contain 
few  or  no  white  corpuscles,  and  do  not  clot  spontaneously,  but  after 
the  addition  of  fibrin  ferment  or  liquids  like  serum  that  contain  fibrin 
ferment  they  always  yield  fibrin. 


THE   BLOOD  109 

Pure  plasma  may  be  obtained  from  horse's  veins  by  what  is  known 
as  the  '  Hving  test-tube  '  experiment.  If  the  jugular  vein  is  ligatured 
in  two  places,  so  as  to  include  a  quantity  of  blood  within  it,  then 
removed  from  the  animal  and  hung  in  a  cool  place,  the  blood  will  not 
coagulate  for  many  hours.  The  corpuscles  settle,  and  the  supernatant 
plasma  can  be  removed  with  a  pipette. 

The  plasma  is  alkaline,  yellowish  in  tint,  and  its  specific  gravity 
is  about  1,026  to  1,029. 

Its  chief  constituents  may  be  enumerated  as  follows  : — 

1,000  parts  of  plasma  contain — 

Water        .         .         .         .         .         .         .  902-90 

Solids         .         .         .         .         .         .  97-10 

Proteins :  1,  yield  of  fibrin         .                  .  4-05 

2,  other  proteins                 .         .  78-84 

Extractives  (including  fat)          .         .         .  5-66 

Inorganic  salts 8*55 

In  round  numbers  plasma  contains  10  per  cent,  of  solids,  of  which 
8  per  cent,  are  protein  in  nature. 

Serum  contains  the  same  three  classes  of  constituents — proteins, 
extractives,  and  salts.  The  extractives  and  salts  are  the  same  in  the 
two  liquids.  The  proteins  differ,  as  is  shown  in  the  following 
table  : — 

Proteins  of  Pldsma  \  Proteins  of  Serum 

Fibrinogen  Serum  globulin 

Serum  globulin  Serum  albumin 

Serum  albumin  Fibrin  ferment  (nucleo- protein) 

The  gases  of  the  plasma  and  serum  are  small  quantities  of 
oxygen,  nitrogen,  and  carbonic  acid.  .  The  greater  part  of  the  oxygen 
of  the  blood  is  combined  in  the  red  corpuscles  with  haemoglobin  ; 
the  carbonic  acid  is  chiefly  combined  as  carbonates  (see  Eespieation). 

We  may  now  consider  one  by  one  the  various  constituents  of  the 
plasma  and  serum. 

A.  Proteins. — Fibrinogen. — This  is  the  parent  substance  of  fibrin, 
It  is  a  globuHn.  It  differs  from  serum  globulin,  and  may  be  sepa- 
rated from  it  by  the  fact  that  half  saturation  with  sodium  chloride 
precipitates  it.  It  coagulates  by  heat  at  the  low  temperature  of 
56°  0.  As  judged  from  the  yield  of  fibrin,  it  is  the  least  abundant  of 
the  proteins  of  the  plasma  (see  table  on  upper  part  of  this  page). 

Serum  globulin  and  serum  albumin. — These  substances  are  con- 
sidered in  the  practical  exercises  at  the  head  of  this  lesson  ;  see  also 


110 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOaY 


Lesson  IV.     Both  serum  globulin  and  serum  albumin  probably  con- 
sist of  more  than  one  protein  substance  (see  Lesson  XX.). 

Fibrin  ferment. — Schmidt's  method  of  preparing  it  is  to  take  serum 
and  add, excess  of  alcohol.  This  precipitates  all  the  proteins,  fibrin 
ferment  included.  After  some  weeks  the  alcohol  is  poured  off ;  the 
serum  globulin  and  serum  albumin  have  been  by  this  means  rendered 
insoluble  in  water ;  an  aqueous  extract  is,  however,  found  to  contain 
fibrin  ferment,  which  is  not  so  easily  coagulated  by  alcohol  as 
the  other  proteins  are.  A  simpler  method  of  preparing  fibrin 
ferment  in  an  impure  but  efficient  form  is  given  in  the  footnote 
on  p.  101. 

B.  Extractives. — These  are  non-nitrogenous  and  nitrogenous.  The 
non-nitrogenous  are  sugar  (0*12  per  cent.),  fats,  soaps,  cholesterin  ;  and 
the  nitrogenous  are  urea  (0*02  to  O'Od  per  cent.),  and  still  smaller 
quantities  of  uric  acid,  creatine,  creatinine,  xanthine,  and  hypoxan- 
thine. 

C.  Salts. — The  most  abundant  salt  is  sodium  chloride  :  it  con- 
stitutes between  60  and  90  per  cent,  of  the  total  mineral  matter. 
Potassium  chloride  is  present  in  much  smaller  amount.  It  consti- 
tutes about  4  per  cent,  of  the  total  ash.  The  other  salts  are  phos- 
phates and  sulphates. 

Schmidt  gives  the  following  table  : — 


1,000  parts  of  plasma  yield — 

Mineral  matter      .         .         .         .         .    •     .         8*550 

Chlorine 

3-640 

SO3         . 

0-115 

P2O5       .         .         . 

0*191 

Potassium 

0*323 

Sodium . 

3*841 

Calcium  phosphate 

0*311 

Magnesium  phosphate 0*222 

THE   WHITE 

BLO( 

)D   ( 

:!ORPTJSC 

LES 

These  corpuscles  are  typical  animal  cells.  Their  nucleus  consists 
of  nuclein  ;  their  cell-protoplasm  yields  proteins  belonging  to  the 
nucleo-protein  and  globulin  groups.  The  protoplasm  of  these  cells 
often  contains  small  quantities  of  fat  and  glycogen. 


THE   RED   BLOOD   CORPUSCLES 

The  red  blood  corpuscles  are  much  more  numerous  than  the  white, 
averaging  in  man  5,000,000  per  cubic  millimetre,  or  400  to  500  red  to 
each  white  corpuscle.  The  method  of  enumeration  of  the  corpuscles 
is  described  in  the  Appendix. 


THE  BLOOD  111 

They  vary  in  size  and  structure  in  different  groups  of  vertebrates. 
In  mammals  they  are  biconcave  (except  in  the  camel  tribe,  where 
they  are  biconvex)  non-nucleated  discs,  in  man  averaging  -j^o  i^^ch 
in  diameter  ;  during  foetal  life  nucleated  red  corpuscles  are,  however, 
found.  In  birds,  reptiles,  amphibians,  and  fishes  they  are  biconvex 
oval  discs  with  a  nucleus  :  they  are  largest  in  the  amphibia. 

Water  causes  the  corpuscles  to  swell  up,  and  dissolves  out  the 
red  pigment  (oxyhaemoglobin),  leaving  a  globular  colourless  stroma. 
Salt    solution    causes     the    corpuscles    ^  , 

shrink  :  they  become  crenated  or  wrinkled.  §  f|  ^  #^  /^ 
The  action  of  water  and  salt  solution  is  8  ©  fir  ^'^  ^-^ 
explained  by  the  existence  of  a  membrane 


on  the  surface  of   the  corpuscles  through    •''-^^  ^C^ 

which  osmosis  takes  place.     Dilute  alkalis        ^^  V^ 

.      7  N      T         1           - 1  ^^^-  29.— a-^,  successive  effects  of 

(0"2     per    cent,     potash)     dissolve     the    cor-  water  on  a  red  blood  corpuscle : 

,              -r^.y     ,            -7/1                        X             J.'  /,  a  red  corpuscle  crenated  by  salt 

pUSCleS.       JJimte    acids    (1    per    cent,    acetic  solution  ;  ,<7,  action  of  tannin  on 

acid)  act  like  water,  and  in  nucleated  cor-  ^^  corpusc  e. 
puscles  render  the  nucleus  distinct.  Tannic  acid  causes  a  discharge 
of  haemoglobin  from  the  stroma,  but  this  is  immediately  altered  and 
precipitated.  It  remains  adherent  to  the  stroma  as  a  brown  globule, 
consisting  probably  of  haematin.  Boric  acid  acts  similarly,  but  in 
nucleated  red  corpuscles  the  pigment  collects  chiefly  round  the 
nucleus,  which  may  then  be  extruded  from  the  corpuscles. 
Composition. — 1,000  parts  of  red  corpuscles  contain — 

Water  .....     688       parts 

Solids       'o>-g^°i«.   •  ■  •  •     303-88  „ 

(inorganic  .  .  .         8*12  „ 
100  parts  of  dried  corpuscles  contain — 

Protein          .        ~     .  .  .         .  .     5  to  12  parts 

Haemoglobin              .  .  .  .  86  ,,  94  ,, 

Lecithin        .             .  .  .  .1*8  part 

Cholesterin   .             .  .  .  .0*1  ,, 

The  protein  present  a,ppears  to  be  identical  with  the  nucleo- 
protein  of  white  corpuscles.  The  mineral  matter  consists  chiefly  of 
chlorides  of  potassium  and  sodium,  and  phosphates  of  calcium  and 
magnesium.  In  man  potassium  chloride  is  more  abundant  than 
sodium  chloride ;  this,  however,  does  not  hold  good  for  all  animals. 

Oxygen  is  contained  in  combination  with  the  haemoglobin  to  form 
oxyhaemoglobin.  The  corpuscles  also  contain  a  certain  amount  of 
carbonic  acid  (see  Eespieation,  at  the  end  of  this  lesson). 

The  pigment  of  the  red  corpuscles. — The  pigment  is  by  far  the 


112 


ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 


most  abundant  and  important  of  the  constituents  of  the  red  cor- 
puscles. It  differs  from  most  other  proteins  in  containing  the  element 
iron  ;  it  is  also  readily  crystallisable. 

It  exists  in  the  blood  in  two  conditions :  in  arterial  blood  it  is 
combined  loosely  with  oxygen,  is  of  a  bright  red  colour,  and  is  called 
oxyhgemoglobin  ;  the  other  condition  is  the  deoxygenated  or  reduced 
haemoglobin  (better  called  simply  haemoglobin).  This  is  found  in  the 
blood  after  asphyxia.  It  also  occurs  in  all  venous  blood — that  is, 
blood  which  is  returning  to  the  heart  after  it*  has  supplied  the  tissues 
with  oxygen.  Venous  blood,  however,  always  contains  a  considerable 
quantity  of  oxyhaemoglobin  also.  Haemoglobin  is  the  oxygen-carrier 
of  the  body,  and  it  may  be  called  a  respiratory  pigment. 

Crystals  of  oxyhaemoglobin  may  be  obtained  with  readiness  from 
the  blood  of  such  animals  as  the  rat,  guinea-pig,  or  dog;  with  diffi- 
culty from  other  animals,  such  as  man, 
ape,  and  most  of  the  common  mam- 
mals. The  following  methods  are  the 
best : — 

1.  Mix  a  drop  of  defibrinated 
blood  of  the  rat  on  a  slide  with  a 
drop  of  water  ;  put  on  a  cover  glass  ; 
in  a  few  minutes  the  corpuscles  are 
rendered  colourless,  and  then  the 
oxyhaemoglobin  crystallises  out  from 
the  solution  so  formed. 

2.  Microscopical  preparations  may 
also  be  made  by  Stein's  method,  which 
consists  in  using  Canada  balsam  in- 
stead of  water  in  the  above  experi- 
ment. 

3.  On  a  larger  scale  the  crystals 
may  be  obtained  by  shaking  the  blood 
with  one-sixteenth  of   its  volume  of 

ether ;  the  corpuscles  dissolve  and  the  blood  assumes  a  laky  appear- 
ance. After  a  period,  varying  from  a  few  minutes  to  days,  abundant 
crystals  are  deposited. 

The  accompanying  figures  represent  the  form  of  the  crystals  so 
obtained. 

In  nearly  all  animals  the  crystals  are  rhombic  prisms ;  but  in  the 
guinea-pig  they  are  rhombic  tetrahedra  (four-sided  pyramids) ;  in  the 
squirrel,  hexagonal  plates ;  and  in  the  hamster,  rhombohedra  and 
hexagonal  plates. 


Fig.  30.— Oxyhaemoglobiu  crystals  magni- 
fied :  1,  from  human  blood  ;  2,  from  the 
guinea-pig  ;  3,  squirrel ;  4,  hamster. 


THE   BLOOD  113 

The  crystals  also  contain  a  varying  amount  of  water  of  crystallisa- 
tion :  this  may  in  part  explain  their  different  crystalline  forms  and 
solubilities.  Different  observers  have  analysed  haemoglobin.  They 
find  carbon,  hydrogen,  nitrogen,  oxygen,  sulphur,  and  iron.  The 
percentage  of  iron  is  0'4.  Oxyhaemoglobin  may  be  estimated  in  the 
blood  (1)  by  the  amount  of  iron  in  the  ash,  or  (2)  by  certain  colori- 
metric  methods  w^hich  are  described  in  the  Appendix. 

Haemoglobin  is  a  conjugated  protein  (see  p.  44),  and  on  the  addi- 
tion of  an  acid  or  alkali  it  is  broken  up  into  two  parts,  a  protein  called 
(jlobin,  and  a  brown  pigment  called  hcematin,  which  contains  all  the 
iron  of  the  original  substance. 

Globin  is  coagulable  by  heat,  soluble  in  dilute  acids,  and  preci- 
pitable  from  such  solutions*  by  ammonia.  It  is  a  member  of  the 
group  of  proteins  called  histories  (see  p.  42). 

Haematin  is  not  crytallisable :  according  to  Nencki  and  Sieber 
its  formula  is  C.32H32N404Fe.  It  presents  different  spectroscopic 
appearances  in  acid  and  alkaline  solutions,  and  yields  several  products 
under  the  influence  of  certain  reagents,  which  we  shall  consider  in 
the  advanced  course.  For  the  present,  we  shall  mention  only  three 
of  these,  hsemin,  hgematoporphyrin,  and  hgemopyrrol. 

Hsemin  is  of  great  importance,  as  the  obtaining  of  this  substance 
in  a  crystalline  form  is  the  best  chemical  test  for  blood.  Haemin 
crystals,  sometimes   called    Teich- 

mann's  crystals,  are  prepared  for  SLT^'         ^^    ^. 

microscopic  examination  by  boiling  y^'J^-^/r       v,     ,^    \ 

a  fragment  of  dried  blood  with  a  f^    ^     ^"^      j  ^=<^ 

drop  of   glacial  acetic   acid   on   a  ^  ^^    *''   \c^  ^  \j     ^>^ 

sHde ;     on     coohng,    dark  -  brown         v    ^^^  |  X   "  ^    ^ 

plates  and  prisms  belonging  to  the      S"!  ^^^     >  /:;l 

triclinic    system,    often    in     star-  ,    ^'^   ^  r       '^w  ^  ,     ]^ji^ 

shaped  clusters  and  with  rounded  *^'       CiJ  "^  /^^     "** 

angles  (fig.  31),  separate  out.  "^         ^    ^  '^  % 

In   the  case  of  an   old  blood-  -rlr     -|  /■  -^^ '' 

stain     it     is     necessary    to      add     a        Fig.  31.— Hajmiu  crystals  magulfied.  (Preyer.) 

crystal  of  sodium  chloride.     Fresh 

blood  contains  sufiftcient  sodium  chloride  in  itself.  The  action  of  the 
acetic  acid  is  (1)  to  split  the  haemoglobin  into  haematin  and  globin ; 
and  (2)  to  evolve  hydrochloric "  acid  from  the  sodium  chloride.  The 
haematin  unites  with  the  hydrochloric  acid,  and  thus  haemin  is  formed. 
Nencki  has  further  shown  that,  when  prepared  in  this  way,  haemin 
also  contains  the  acetyl  gi'oup. 

Haematoporphyrin  is  iron-free  haematin  :  it  may  be  prepared  by 

I 


114  ESSENTIALS   OF  CHEMICAL  PHYSIOLOaY 

mixing  blood  with  strong  sulphuric  acid  ;  the  iron  is  taken  out  as 
ferrous  sulphate.  This  substance  is  also  found  sometimes  in  nature  ; 
it  occurs  in  certain  invertebrate  pigments,  and  may  also  be  found  in 
certain  forms  of  pathological  urine.  It  shows  well-marked  spectro- 
scopic bands,  and  so  is  not  identical  with  the  iron-free  derivative  of 
haemoglobin  called  haematoidin  which  is  formed  in  extravasations  of 
blood  in  the  body  (see  p.  90).  The  two  substances  are  possibly 
isomeric. 

Haemopyrrol  is  methyl-propyl-pyrrol,  with  the  formula 

H.C-C  — CH,.C,H5 

II      II 
H.C     G  — CH3 


N.H 

and  is  obtained  by  reduction  from  haematoporphyrin.  Ifc  is  also 
similarly  obtained  from  the  derivative  of  chlorophyll  called  phyllo- 
porphyrin,  a  fact  which  illustrates  the  near  relationship  of  the 
principal  animal  and  vegetable  pigments. 

COMPOUNDS   OF  H.ffiMOGLOBIN   WITH  GASES 

Haemoglobin  forms  at  least  four  compounds  with  gases  : — 

^T-,,  (    1.  Oxyhaemoglobin. 

With  oxygen  i    r.    ^r  .^  -.  ,  • 

•^^  12.  Methaemoglobm. 

With  carbonic  oxide  3.  Carbonic  oxide  haemoglobin. 

With  nitric  acid  4.  Nitric  oxide  haemoglobin. 

These  compounds  have  similar  crystalline  forms  :  each  probably 
consists  of  a  molecule  of  haemoglobin  combined  with  one  of  the  gas- 
They  part  with  the  combined  gas  somewhat  readily,  and  are  arranged 
in  order  of  stability  in  the  above  list,  the  least  stable  first. 

Oxyhaemoglobiii  is  the  compound  that  exists  in  arterial  blood.  The 
oxygen  linked  to  the  haemoglobin,  which  is  removed  by  the  tissues 
through  which  the  blood  circulates,  may  be  called  the  resjnratory 
oxygen  of  hcevwglobin.  The  processes  that  occur  in  the  lungs  and 
tissues,  resulting  in  the  oxygenation  and  deoxygenation  respectively 
of  the  haemoglobin,  may  be  imitated  outside  the  body,  using  either 
blood  or  pure  solutions  of  haemoglobin.  The  respiratory  oxygen  can 
be  removed,  for  example,  in  the  Torricellian  vacuum  of  a  mercurial 
air-pump,  or  by  passing  a  neutral  gas  like  hydrogen  through  the 
blood  or  by  the  use  of  reducing  agents  such  as  ammonium  sulphide  or 


THE  BLOOD  115 

Stokes's  reagent.^  One  gramme  of  haemoglobin  will  combine  with 
1-34:  c.c.  of  oxygen. 

If  any  of  these  methods  for  reducing  oxyhaemoglobin  is  used,  the 
bright  red  (arterial)  colour  of  oxyhaemoglobin  changes  to  the  purpHsh 
(venous)  tint  of  haemoglobin.  On  once  more  allowing  oxygen  to 
come  into  contact  with  the  haemoglobin,  as  by  shaking  the  solution 
with  the  air  the  bright  arterial  colour  returns. 

These  colour-changes  may  be  more  accurately  studied  with  the 
spectroscope,  and  the  constant  position  of  the  absorption  bands  seen 
constitutes  an  important  test  for  blood  pigment. 

The  Spectroscope. — "When  a  ray  of  white  Hght  is  passed  through 
a  prism,  it  is  refracted  or  bent  at  each  surface  of  the  prism ;  the 
whole  ray  is,  however,  not  equally  bent,  but  it  is  spht  into  its  con- 
stituent colours,  which  may  be  allowed  to  fall  on  a  screen.  The 
band  of  colours  beginning  with  the  red,  passing  through  orange, 
yellow,  green,  blue,  and  ending  with  violet,  is  called  a  spectrum  :  this 
is  seen  in  nature  in  the  rainbow. 

The  spectrum  of  sunlight  is  interrupted  by  numerous  dark  lines 

crossing  it  vertically  called  Fraunhofer's  lines.     These  are  perfectly 

constant   in    position,    and   serve   as   landmarks   in    the    spectrum. 

The  more  prominent  are  A,  B,  and  0,  in  the  red  ;  D,  in  the  yellow ; 

E,  b,  and  F,  in  the  green ;  G  and  H,  in  the  violet.     These  lines  are 

due  to  certain  volatile  substances  in  the  solar  atmosphere.     If  the 

light  from  burning  sodium  or  its  compounds  is  examined  spectro- 

scopically,  it  will  be  found  to  give  a  bright  yellow  line,  or  rather 

two  bright  yellow  lines  very  close  together.     Potassium  gives  two 

bright  red  Hues  and  one  violet  line ;  and  the  other  elements,  when 

incandescent,  give  characteristic  lines,  but  none  so  simple  as  sodium. 

If  now  the  flame  of  a  lainp  be  examined,  it  will  be  found  to  give  a 

continuous  spectrum  like  that  of  sunlight  in  the  arrangement  of  its 

colours,  but  unlike  it  in  the  absence  of  dark  lines  ;  but  if  the  light 

from  the  lamp  be  made  to  pass  through  sodium  vapour  before  it 

reaches    the    spectroscope,    the   brighb   yellow  light  will   be   found 

absent,  and  in  its  place  a  dark  line,  or  rather  two  dark  lines  very 

close  together,  occupying  the  same  position  as  the  two  bright  hues 

of  the  sodium  spectrum.     The  sodium  vapour  thus  absorbs  the  same 

rays  as  those  which  it  itself  produces  at  a  higher  temperature.     Thus 

the  D  line,  as  we  term  it,  in  the  solar  spectrum  is  due  to  the  presence 

of  sodium  vapour  in  the  solar  atmosphere.     The  other  dark  lines  are 

similarly  accounted  for  by  other  elements. 

*  Stokes's  reagent  must  always  be  freshly  prepared  :  it  is  a  solution  of  ferrous 
sulphate  to  which  a  little  tartaric  acid  has  been  added,  and  then  ammonia  till  the 
reaction  is  alkaline. 

I  2 


116 


ESSENTIALS  OF  CHEMICAL   PHYSIOLOGY 


The  large  form  of  spectroscope  (fig.  32)  consists  of  a  tube  A,  called 
the  collimator,  with  a  slit  at  the  end  S,  and  a  convex  lens  at  the  end  L. 
The  latter  makes  the  rays  of  light  passing  through  the  slit  from  the 
source  of  light  parallel :  they  fall  on  the  prism  P,  and  then  the 
spectrum  so  formed  is  focussed  by  the  telescope  T. 


Fig.  32. — Diagniin  of  aptctroscope. 

The  third  tube,  D,  seen  in  the  next  figure  (fig.  33)  carries  a  small 
transparent  scale  of  wave-lengths,  so  that  the  position  of  any 
point  in  the  spectrum  may  be  given  in  terms  of  the  corresponding 
wave-lengths. 


Fig.  33. — Spectroscope:  A,  collimator  with  adinstablc  slit  at  one  (left)  end  and  collimating  lens  at 
the  other  (right)  end  ;  B,  telei-c(Ji)e  moving  on  graduated  arc  divided  into  degrees  ;  C,  prism  or 
combination  of  prisms ;  D,  tube  for  scale ;  E,  miiTor  for  illuminating  scale ;  F,  vessel  with 
parallel  glass  sides  for  holding  lluid,  shown  with  the  flat  ?ide  towards  the  reader  ;  I,  long 
spectroscope  bottle  for  examining  a  deep  layer  of  fluid  ;  H,  Argaud  burner  ;  G,  condenser  for 
concentrating  the  light  from  H  on  the  slit,  (From  a  photograph  taken  by  Dr.MacMunii  for 
McKendrick's  '  Physiologj'.') 

If  we  now  interpose  between  the  source  of  light  and  the  slit  S 
a  piece  of  coloured  glass  (H  in  fig.  32),  or  a  solution  of  a  coloured 


THE   BLOOD  117 

substance  contained  in  a  vessel  with  parallel  sides  (the  haematoscope 
of  Hermann,  F  in  fig.  33),  the  spectrum  is  found  to  be  no  longer 
continuous,  but  is  interrupted  by  a  number  of  dark  shadows,  or 
absoyytion  hands,  corresponding  to  the  light  absorbed  by  the  coloured 
medium.  Thus  a  solution  of  oxyhaemoglobin  of  a  certain  strength 
gives  two  bands  between  the  D  and  E  lines  ;  haemoglobin  gives  only 
one  ;  and  other  red  solutions,  though  to  the  naked  eye  similar  to 
oxyhaemoglobin,  will  give  characteristic  bands  in  other  positions. 

A  convenient  form  of  small  spectroscope  is  the  direct-vision 
sioectroscoye,  in  which,  by  an  arrangement  of  alternating  prisms 
of  crown  and  flint  glass  (see  fig.  34),  the  spectrum  is  observed 
by  the  eye  in  the  same  line  as  the  tube  furnished  with  the  slit. 
Such  small  spectroscopes  may  be  used  for  class  purposes,  and 
may  for  convenience  be  mounted  on  a  stand  provided  with  a  gas- 
burner  and  a  receptacle  for  the  test-tube  (see  fig.  35).  In  the 
examination  of  the  spectrum  of  small  coloured  objects,  a  combina- 
tion of  the  microscope  and  direct-vision  spectroscope,  called  the 
micro- spectroscope,  is  used. 

Fig.  36  illustrates  a  method  of  representing  absorption  spectra 


Fig.  34.— Arrangrement  of  prisms  in  direct-vision  spectroscope. 

diagrammatically.  The'  solution'  was  examined  in  a  layer  1  cen- 
timetre thick.  The  base  line  has  on  it  at  the  proper  distances 
the  chief  Fraunhofer  lines,  and  along  the  right-hand  edges  are 
the  percentage  amounts  of  oxyhaemoglobin  present  in  I,  of  haemo- 
globin in  II.  The  width  of  the  shadings  at  each  level  represents  the 
position  and  amount  of  absorption  corresponding  to  the  percentages. 
The  characteristic  spectrum  of  oxyhaemoglobin,  as  it  actually 
appears  through  the  spectroscope,  is  seen  in  the  next  figure  (fig.  37^ 
spectrum  2).  There  are  two  distinct  absorption  bands  between  the 
D  and  E  lines  ;  the  one  nearest  to  D  (the  a  band)  is  narrower, 
darker,  and  has  better  defined  edges  than  the  other  (the  /3  band). 
As  will  be  seen  on  looking  at  fig.  36,  a  solution  of  oxyhaemoglobin  of 
concentration  greater  than  0-65  per  cent,  and  less  than  0-85  per  cent., 
(examined  in  a  cell  of  the  usual  thickness  of  1  centimetre)  gives  one 


118 


ESSENTIALS   OF  CHEMICAL  PHYSIOLOaY 


thick  band  overlapping  both  D  and  E,  and  a  stronger  solution  only 
lets  the  red  light  through  between  C  and  D.     A  solution  which  gives 


Fig.  35.— Stand  for  direct-vision  spectroscope  :  S,  spectroscope ;  T,  test-tnbe  for  coloured 
substance  under  investigation. 

the  two  characteristic  bands  must  therefore  be  a  very  dilute  one. 
The  one  band  (y  band)  of  haemoglobin  (fig.  37,  spectrum  3)  is  not  so 


Fig.  36.— Graphic  representations  of  the  amount  of  absorption  of  light  by  solution  (I)  of  oxyhaemo- 
globin,  (IT)  of  haemoglobin,  of  different  strengtlis.  The  shading  indicates  the  amount  of 
absorption  of  the  spectrum  ;  the  figures  on  the  right  border  express  percentages.    (Rollett.) 

well  defined  as  the  a  and  p  bands.     On  dilution  it  fades  rapidly,  so 
that  in  a  solution  of  such  strength  that  both  bands  of  oxyhaemoglobin 


THE   ELOOD 


119 


would  be  quite  distinct  the  single  band  of  haemoglobin  has  dis- 
appeared from  view.  The  oxyhaemoglobin  bands  can  be  distinguished 
in  a  solution  which  contains  only  one  part  of  the  pigment  to  10,000 
of  water,  and  even  in  more  dilute  solutions  which  seem  to  be  colourless 
the  a  band  is  still  visible. 

Methaemoglobm. — This  may  be  produced  artificially  by  adding 
such  reagents  as  potassium  ferricyanide  or  amyl  nitrite  to  a  solution 
of  oxyhaemoglobin  ;  it  may  also  occur  in  certain  diseased  conditions 
in  the  urine  ;  it  is  therefore  of  considerable  practical  importance.  It 
can  be  crystallised,  and  is  found  to  contain  the  same  amount  of 
oxygen  as  oxyhaemoglobin,  only  combined  differently.  The  oxygen 
is  not  removable  by  the  air-pump,  nor  by  a  stream  of  a  neutral  gas 
C         D  E&  F  G 


Fig.  37,-1,  Solar  spectrum  :  '2.  ^p.  Mrrum  of  oxyhaemoglobin  (0-37  per  cent,  solution)  ;  3,  spectrum  of 
hasmoglobin  ;  4,  spectrum  of  CO-h«moglobin  ;  5,  spectrum  of  methfemoglobin  (concentrated 
solution). 

like  hydrogen.  It  can,  however,  by  reducing  agents  like  ammonium 
sulphide,  be  made  to  yield  haemoglobin.  Methaemoglobin  is  of  a 
brownish-red  colour,  and  gives  a  characteristic  absorption  band  in 
the  red  between  the  C  and  D  lines  (fig.  37,  spectrum  5). 

Tho  ferricyanide  of  potassium  or  sodium  not  only  causes  the 
conversion  of  oxyhaemoglobin  into  methaemoglobin,  but  if  the  reagent 
is  added  to  blood  which  has  been  previously  laked  by  the  addition  of 
twice  its  volume  of  water  there  is  an  evolution  of  oxygen.  If  a  small 
amount  of  sodium  carbonate  or  ammonia  is  added  as  well  to  prevent 
the  evolution  of  any  carbonic  acid,  and  the  oxygen  is  collected  and 
measured,  it  is  found  that  all  the  oxygen  previously  combined  in 
oxyhaemoglobin  is  discharged.    This  is  at  first  sight  puzzling,  because, 


120  ESSENTIALS  OF  CHEMICAL  PHYSIOLOOY 

as  just  stated,  methacmoglobin  contains  the  same  amount  of  oxygen 
that  is  present  in  oxyhaBmoglobin.  What  occurs  is  that,  after  the 
oxygen  is  discharged  from  oxyhaemoglobin,  an  equal  quantity  of 
oxygen  takes  its  place  from  the  reagents  added.  The  oxygen  atoms 
of  the  methsemoglobin  must  be  attached  to  a  different  part  of  the 
haematin  group  from  the  oxygen  atoms  of  the  oxyhaemoglobin,  so 
that  the  haematin  group  when  thus  altered  loses  its  power  of  com- 
bining with  oxygen  and  carbonic  oxide  to  form  compounds  which 
are  dissociable  in  a  vacuum. 

Haldane,  to  whom  we  owe  these  interesting  results,  gives  the 
following  provisional  equation  to  represent  what  occurs  : — 

Hb02  +  4Na3CycFe  +  4NaHCO,=Hb02  +  4Na4Cy,Fe 

[oxyhaemo-       [sodium  ferri-        [sodium  bicar-      [metlifemo-      [sodium  ferro- 
globin]  cyanide]  bnnate]  globin]  cyanide] 

+  4CO2  +  P.H2O  +  O2. 

[carbonic     [water]        [oxygen] 
acid] 

Carbonic  Oxide  Haemoglobin  may  be  readily  prepared  by  passing 
a  stream  of  carbonic  oxide  or  coal  gas  through  blood  or  through  a 
solution  of  oxyhaemoglobin.  It  has  a  peculiar  cherry-red  colour.  Its 
absorption  spectrum  is  very  like  that  of  oxyhaemoglobin,  but  the  two 
bands  are  slightly  nearer  the  violet  end  of  the  spectrum  (fig.  37, 
spectrum  4).  Keducing  agents,  such  as  ammonium  sulphide,  do  not 
change  it ;  the  gas  is  more  firmly  combined  than  the  oxygen  in 
oxyhaemoglobin.  CO-haemoglobin  forms  crystals  like  those  of  oxy- 
haemoglobin :  it  resists  putrefaction  for  a  very  long  time. 

Carbonic  oxide  is  given  off  during  the  imperfect  combustion  of 
carbon  such  as  occurs  in  charcoal  stoves  ;  it  is  a  powerful  poison 
combining  with  the  haemoglobin  of  the  blood,  and  thus  interfering 
with  normal  respiratory  processes.  The  colour  of  the  ])lood  and  its 
resistance  to  reducing  agents  are  in  such  cases  characteristic.  . 

Nitric  Oxide  Haemoglobin. — When  ammonia  is  added  to  blood,  and 
then  a  stream  of  nitric  oxide  is  passed  through  it,  this  compound  is 
formed.  It  may  be  obtained  in  crystals  isomorphous  with  oxy-  and 
CO-haemoglobin.  It  also  has  a  similar  spectrum.  It  is  even  more 
stable  than  CO-haemoglobin ;  it  has  little  practical  interest,  but  is  of 
theoretical  importance  as  completing  the  series. 

TESTS   FOR  BLOOD 

These  may  be  gathered  from  preceding  descriptions.  Briefly, 
they  are  microscopic,  spectroscopic,  and  chemical.  The  best  chemical 
test  is  the  formation  of  haemin  crystals.     The  old  test  with  tincture 


THE   BLOOD  121 

of  guaiacum  and  hydrogen  peroxide,  the  blood  causing  the  red  tincture 
to  become  green,  is  not  very  trustworthy,  as  it  is  also  given  by  many 
other  organic  substances ;  it  is,  however,  with  certain  precautions, 
used  by  some. 

In  medico-legal  cases  it  is  often  necessary  to  ascertain  whether 
a  red  fluid  or  stain  upon  clothing  is  or  is  not  blood.  In  any  such 
case  it  is  advisable  not  to  rely  upon  one  test  only,  but  to  try  every 
means  of  detection  at  one's  disposal.  To  discover  whether  it  is  blood 
or  not,  is  by  no  means  a  difficult  problem,  but  to  distinguish  human 
blood  from  that  of  the  common  mammals  is  possible  only  by  the 
*  biological '  test  described  at  the  end  of  the  next  section. 

IMMUNITY 

The  chemical  defences  of  the  body  against  injury  and  disease 
are  numerous.  The  property  that  the  blood  possesses  of  coagu- 
lating is  a  defence  against  haemorrhage  ;  the  acid  of  the  gastric 
juice  is  a  protection  against  harmful  bticteria  introduced  with  food. 
Bacterial  activity  in  urine  is  inhibited  by  the  acidity  of  that  secretion. 

Far  more  important  and  widespread  in  its  effects  than  any  of  the 
foregoing  is  the  bactericidal  {i.e.  bacteria-killing)  action  of  the  blood 
and  lymph  ;  a  study  of  this  question  has  led  to  many  interesting 
results,  especially  in  connection  with  the  important  problem  of 
immunity. 

It  is  a  familiar  fact  that  one  attack  of  many  of  the  infective  maladies 
protects  us  against  another  attack  of  the  same  disease.  The  person 
is  said  to  be  immune,  either  partially  or  completely,  against  that 
disease.  Vaccination  produces  in  a  patient  an  attack  of  cowpox  or 
vaccinia.  This  disease  is  either  closely  related  to  smallpox,  or 
may  be  it  is  smallpox  modified  and  rendered  less  malignant  by  passing 
through  the  body  of  a  calf.  At  any  rate,  an  attack  of  vaccinia  renders 
a  person  immune  to  smallpox  for  a  certain  number  of  years.  Vac- 
cination is  an  instance  of  what  is  called  'protective  inoculation,  which 
is  now  practised  with  more  or  less  success  in  reference  to  other 
diseases,  such  as  plague  and  typhoid  fever.  The  study  of  immunity 
has  also  rendered  possible  what  may  be  called  curative  hwculation,  or 
the  injection  of  antitoxic  material  as  a  cure  for  diphtheria,  tetanus, 
snake  poisoning,  &c. 

The  power  the  blood  possesses  of  slaying  bacteria  is  not  limited 
to  the  colourless  corpuscles  ox  phagocytes,  but  is  also  a  property  of  the 
fluid  part  of  the  blood,  at  any  rate  in  the  case  of  some  micro- 
organisms. The  chemical  characters  of  the  substances  which  kill 
the  bacteria  are  not   fully  known ;    but   they  appear   to   be  protein 


122  ESSENTIALS   OF   CHEMICAL   PHYSIOLOaY 

in  nature.  The  bactericidal  powers  of  blood  are  destroyed  by 
heating  it  for  an  hour  to  55°  C.  The  balance  of  evidence  appears 
to  be  in  favour  of  the  view  that  the  substances  in  question 
originate  from  the  leucocytes,  and  phagocytosis  becomes  more 
intelligible  if  this  is  accepted.  The  substances,  whatever  be  their 
source  or  their  chemical  nature,  are  called  hacterio-lysins. 

Closely  allied  to  the  bactericidal  power  of  blood,  or  blood-serum, 
is  its  globulicidal  power.  By  this  one  means  that  the  blood-serum  of 
one  animal  has  the  power  of  dissolving  the  red  blood-corpuscles  of 
another  species.  If  the  serum  of  one  animal  is  injected  into  the 
blood-stream  of  an  animal  of  another  species,  the  result  is  a  destruction 
of  its  red  corpuscles,  which  may  be  so  excessive  as  to  lead  to  the 
passing  of  the  liberated  haemoglobin  into  the  urine  (haemoglobinuria). 
The  substances  in  the  serum  that  possess  this  property  are  called 
hcemolysins,  and  though  there  is  some  doubt  whether  bacterio- 
lysins  and  haemolysins  are  absolutely  identical,  there  is  no  doubt 
that  they  are  closely  related  substances. 

Normal  blood  thus  possesses  not  only  j^hagocytes,  which  eat  up 
bacteria,  but  also  a  certain  amount  of  chemical  substances  which  are 
inimical  to  the  life  of  our  bacterial  foes.  But  suppose  a  person  gets 
'  run  down '  ;  everyone  knows  he  is  then  more  liable  to  '  catch  any- 
thing.' This  coincides  with  a  diminution  in  the  bactericidal  power  of 
his  blood.  But  even  a  perfectly  healthy  person  has  not  an  unlimited 
supply  of  bacterio-lysins,  and  if  the  bacteria  are  sufficiently  numerous 
he  will  fall  a  victim  to  the  disease  they  produce.  Here,  however, 
comes  in  the  remarkable  part  of  the  defence.  In  the  struggle  he 
will  produce  more  and  more  bacterio-lysin,  and  if  he  gets  well  it 
means  that  the  bacteria  are  finally  vanquished,  and  his  blood  remains 
rich  in  the  particular  bacterio-lysin  he  has  produced,  and  so  will 
render  him  immune  to  further  attacks  from  that  particular  species 
of  bacterium.  Every  bacterium  seems  to  cause  the  development  of 
a  specific  anti -substance. 

Immunity  can  more  conveniently  be  produced  gradually  in  animals, 
and  this  applies,  not  only  to  the  bacteria,  but  also  to  the  toxins  they 
form.  If,  for  instance,  the  bacilli  which  produce  diphtheria  are 
grown  in  a  suitable  medium,  they  produce  the  diphtheria  poison,  or 
toxin,  much  in  the  same  way  that  yeast-cells  will  produce  alcohol 
when  grown  in  a  solution  of  sugar.  Diphtheria  toxin  is  associated 
with  a  proteose,  as  is  also  the  case  with  the  poison  of  snake  venom. 
If  a  certain  small  dose  called  a  '  lethal  dose  '  is  injected  into  a  guinea- 
pig  the  result  is  death.  But  if  the  guinea-pig  receives  a  smaller 
dose  it  will  recover  ;  a  few  days  after  it  will  stand  a  rather  larger 


THE  BLOOD  123 

dose  ;  and  this  maj^  be  continued  until,  after  many  successive  gradually 
increasing  doses,  it  will  finally  stand  an  amount  equal  to  many  lethal 
doses  without  any  ill  effects.  The  gradual  introduction  of  the  toxin 
has  called  forth  the  production  of  an  antitoxin.  If  this  is  done  in 
the  horse  instead  of  the  guinea-pig  the  production  of  antitoxin  is 
still  more  marked,  and  the  serum  obtained  from  the  blood  of  an 
immunised  horse  maybe  used  for  injecting  into  human  beings  suffering 
from  diphtheria,  and  it  rapidly  cures  the  disease.  The  two  actions  of 
the  blood,  antitoxic  and  antibacterial,  are  frequently  associated,  but 
may  be  entirely  distinct. 

The  antitoxin  is  also  a  protein  probably  of  the  nature  of  a  globulin  ; 
at  any  rate  it  is  a  protein  of  larger  molecular  weight  than  a  proteose. 
This  suggests  a  practical  point.  In  the  case  of  snake-poisoning  the 
poison  gets  into  the  blood  rapidly  owing  to  the  comparative  ease  with 
which  it  diffuses,  and  so  it  is  quickly  carried  all  over  the  body.  In 
treatment  with  the  antitoxin  or  antivenin,  speed  is  everything  if  life 
is  to  be  saved  ;  injection  of  this  material  under  the  skin  is  not  much 
good,  for  the  diffusion  into  the  blood  is  too  slow.  It  should  be 
injected  straight  away  into  a  blood-vessel. 

There  is  no  doubt  that  in  these  cases  the  antitoxin  neutralises  the 
toxin  much  in  the  same  way  that  an  acid  neutralises  an  alkali.  If 
the  toxin  and  antitoxin  are  mixed  in  a  test-tube,  and  time  allowed 
for  the  interaction  to  occur,  the  result  is  an  innocuous  mixture.  The 
toxin,  however,  is  merely  neutralised,  not  destroyed  ;  for  if  the  mix- 
ture in  the  test-tube  is  heated  to  68°  C.  the  antitoxin  is  coagulated 
and  destroyed,  and  the  toxin  remains  as  poisonous  as  ever. 

Immunity  is  distinguished  into  active  and  passive.  Active  immunity  is 
produced  by  the  development  of  protective  substances  in  the  body ;  passive 
immunity  by  the  injection  of  a  protective  serum.  Of  the  two  the  former  is 
the  more  permanent. 

Bicin,  the  poisonous  protein  of  castor-oil  seeds,  and  ahriv,  that  of  the 
Jequirity  bean,  also  produce  when  gradually  given  to  animals  an  immunity, 
due  to  the  production  of  antiricin  and  antiabrin  respectively. 

Ehrlich's  hypothesis  to .  explain  such  facts  is  usually  spoken  of  as  the 
Hide-chain  theory  of  imnmnity.  He  considers  that  the  toxins  are  capable  of 
uniting  with  the  protoplasm  of  the  living  cells  by  possessing  groups  of  atoms 
like  those  by  which  nutritive  proteins  are  united  to  cells  during  nonnal 
assimilation.  He  terms  these  haptoj^hor  groups,  and  the  groups  to  which 
these  are  attached  in  the  cells  he  terms  receptor  groups.  The  introduction 
of  a  toxin  stimulates  an  excessive  production  of  receptors,  which  are  finally 
thrown  out  into  the  circulation,  and  the  free  circulating  receptors  constitute 
the  antitoxin.  The  comparison  of  the  process  to  assimilation  is  justified  by 
the  fact  that  non-toxic  substances  like  milkor  egg-white  introduced  gradually 
by  successive  doses  into  the  blood-stream  cause  the  formation  of  anti-sub- 
stances capable  of  coagulating  them. 

Up  to  this  point  I  have  spoken  only  of  the  blood,  but  m.onth  by  month 
workers  are  bringing  forward  evidence  to  show  that  other  cells  of  the  body 


124  ESSENTIALS   OV  CHEMICAL   PHYSIOLOaV 

may  by  similar  measures  be  rendered  capable  of  producing  a  corresponding 
protective  mechanism. 

One  further  development  of  the  theory  I  must  mention.  At  least  two 
different  substances  are  necessary  to  render  a  serum  bactericidal  or  globulicidal. 
The  bacterio-lysin  or  haemolysin  consists  of  these  two  substances.  One  of 
these  is  called  the  immune  body,  the  other  the  comjjlement.  We  may 
illustrate  the  use  of  these  terms  by  an  example.  The  repeated  injection  of 
the  blood  of  one  anhnal  {e.g.  the  goat)  into  the  blood  of  another  animal  {e.g. 
a  sheep)  after  a  time  renders  the  latter  animal  immune  to  farther  injections, 
and  at  the  same  time  causes  the  production  of  a  serum  which  dissolves 
readily  the  red  blood-corpuscles  of  the  first  animal.  The  sheep's  serum  is 
thus  hemolytic  towards  goat's  blood-corpuscles.  This  power  is  destroyed  by 
heating  to  56°  C.  for  half  an  hour,  but  returns  when  fresh  serum  of  any 
animal  is  added.  The  specific  immunising  substance  formed  in  the  sheep 
is  called  the  immune  body ;  the  ferment-like  substance  destroyed  by  heat 
is  the  complement.  The  latter  is  not  specific,  since  it  is  furnished  by  the 
blood  of  non-immunised  animals,  but  it  is  nevertheless  essential  for  haemo- 
lysis. Ehrlich  believes  that  the  immune  body  has  two  side  groups — one 
which  connects  with  the  receptor  of  the  red  corpuscles,  and  one  which  unites 
with  the  haptophor  group  of  the  complement,  and  thus  renders  possible  the 
ferment-like  action  of  the  complement  on  the  red  corpuscles.  Various 
antibacterial  serums,  which  have  not  been  the  success  in  treating  disease  they 
were  expected  to  be.  are  probably  too  poor  in  complement,  though  they  may 
contain  plenty  of  the  immune  bodj'. 

To  put  it  another  way :  the  cell-dissolving  substances  cannot  act  on  their 
objects  of  attack  without  an  intermediate  substance  to  anchor  them  on  the 
substance  in  question.  This  intermediary  substance,  known  as  the  immune 
body  or  amboceptor,  is  specific,  and  varies  with  the  substance  to  be  attacked 
(red  corpuscles,  bacterium,  toxin,  &c.).  The  complement  may  be  compared 
to  a  person  who  wants  to  unlock  a  door ;  to  do  this  effectively  he  must  be 
provided  with  the  proper  key  (amboceptor  or  immune  body). 

Quite  distinct  from  the  bactericidal,  globulicidal,  and  antitoxic 
properties  of  blood  is  its  agglutinating  action.  This  is  another  result 
of  infection  with  many  kinds  of  bacteria  or  their  toxins.  The  blood 
acquires  the  property  of  rendering  immobile  and  clumping  together 
the  specific  bacteria  used  in  the  infection.  The  test  applied  to  the 
blood  in  cases  of  typhoid  fever,  and  generally  called  Widal's  reaction, 
depends  on  this  fact. 

The  substances  that  produce  this  effect  are  called  agglutinins. 
They  also  are  probably  protein-like  in  nature,  but  are  more  resistant 
to  heat  than  the  lysins.  Prolonged  heating  to  over  60°  C.  is  necessary 
to  destroy  their  activity. 

We  thus  see  that  the  means  the  body  possesses  of  combating 
bacterial  invasion  are  numerous.  In  some  cases  the  bacteria  are 
killed  by  bacterio-lysins,  and  in  other  cases  they  are  directly  attacked 
and  devoured  by  the  phagocytes.  Bacteria  which  are  destroyed  in 
this  way  produce  no  evil  results,  whereas  those  which  are  not 
destroyed  are  called  pathogenic,  or  disease-producing  organisms. 
There  is  still  another  means  of  defence,  for  if  the  bacteria  are  not 


THE   13L00D  126 

'destroyed  the  poisons  or  toxins  they  produce  are  in  certain  other 
cases  ueutrahsed  by  antitoxins. 

Metschnikoff's  view,  which  is  very  widely  accepted  by  bacterio- 
logists, is  that  the  most  stress  should  be  laid  upon  phagocytosis  as 
the  principal  factor  in  tha  resistance  of  the  body  to  bacteria ;  and  the 
recent  discovery  of  opsonins  by  Sir  A.  E.  Wright  not  only  emphasises 
this  opinion,  but  shows  how  the  body  fluids  co-operate  with  the 
phagocytes  in  the  process.  The  word  *  opsonin '  is  derived  from  a 
Greek  word  which  means  'to  prepare  the  feast.'  Washed  bacteria 
from  a  culture  are  distasteful  to  leucocytes,  and  would  therefore, 
other  things  being  equal,  be  pathogenic  if  injected  into  an  animal's 
body.  But  if  the  bacteria  have  been  previously  soaked  in  serum, 
especially  if  that  serum  has  been  obtained  from  the  blood  of  an 
animal  previously  immunised  against  that  special  bacterium,  then 
the  leucocytes  devour  them  eagerly.  It  was  at  first  supposed  that 
something  had  been  added  to  the  bacterium  to  make  it  tasty,  and 
that  each  kind  of  bacterium  requires  its  own  special  sauce  or 
opsonin.  It  is,  however,  equally  possible  that  the  serum  has  not 
added  anything  to  the  bacterium,  but  removed  from  it  something 
that  previously  made  it  distasteful.  At  any  rate  the  ultimate  effect 
is  the  same,  and  the  bacterium  is  rendered  non-pathogenic.  When  a 
person  is  attacked  with  some  invading  organism,  say  the  tubercle 
bacillus,  if  that  person's  blood  is  naturally  rich  in  the  proper  kind  of 
opsonin  he  will  not  be  troubled  with  tuberculosis  ;  but  if  the  opsonic 
power  of  his  blood  is  low  the  organism  will  produce  the  disease. 
The  modern  treatment  of  tuberculosis  aims  at  increasing  the  opsonic 
power  of  the  blood  by  improving  the  general  condition  of  the  patient 
by  good  food  and  pure  air,  and  also  by  the  injection  of  the  appro- 
priate opsonin  into  his  -blood. 

Lastly,  we  come  to  a  question  which  more  directly  appeals  to  the 
physiologist  than  the  preceding,  because  experiments  in  relation  to 
immunity  have  furnished  us  with  what  has  hitherto  been  lacking, 
a  means  of  distinguishing  human  blood  from  the  blood  of  other 
animals. 

The  discovery  was  made  by  Tchistovitch  (1899),  and  his  original 
experiment  was  as  follows  : — Eabbits,  dogs,  goats,  and  guinea-pigs 
were  inoculated  with  eel-serum,  which  is  toxic ;  he  thereby  obtained 
from  these  animals  an  antitoxic  serum.  But  the  serum  was  not  only 
antitoxic  ;  it  also  produced  a  precipitate  when  added  to  eel-serum, 
though  not  when  added  to  the  serum  of  any  other  animal.  In  other 
w^ords,  not  only  has  a  specific  antitoxin  been  produced,  but  also  a 
specific  precipitin.     Numerous  observers  have  since  found  that  this  is 


126 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


a  general  rule  throughout  the  animal  kingdom,  including  man.  If,  for 
instance,  a  rabbit  is  treated  with  human  blood,  the  serum  ultimately 
obtained  from  the  rabbit  contains  a  specific  precipitin  for  human 
blood  ;  that  is  to  say,  a  precipitate  is  formed  on  adding  such  a  rabbit's 
serum  to  human  blood,  but  not  when  added  to  the  blood  of  any  other 
animal.^  The  great  value  of  the  test  is  its  delicacy:  it  will  detect 
the  specific  blood  when  it  is  greatly  diluted,  after  it  has  been  dried 
for  weeks,  or  even  when  it  is  mixed  with  the  blcod  of  other  animals. 

CHEMISTRY  OF   RESPIRATION 

The  consideration  of  the  blood,  and  especially  of  its  pigment,  is  so 
closely  associated  with  respiration  that  a  brief  account  of  that  process 
follows  conveniently  here. 

The  air  in  the  alveoli  of  the  lungs,  and  the  blood  in  the  pulmonary 
capillaries  are  only  separated  by  the  thin  capillary  and  alveolar 
walls.  The  blood  parts  with  its  excess  of  carbonic  acid  and  watery 
vapour  to  the  alveolar  air ;  the  blood  at  the  same  time  receives  from 
the  alveolar  air  the  oxygen  which  renders  it  arterial. 

The  intake  of  oxygen  is  the  commencement,  and  the  output  of 
carbonic  acid  the  end,  of  the  series  of  changes  known  as  respiration. 
The  intermediate  steps  take  place  all  over  the  body,  and  constitute 
what  is  known  as  internal  or  tissue  respiration.  The  exchange  of 
gases  which  occurs  in  the  lungs  is  sometimes  called  in  contradistinc- 
tion exterjial  respiration.  We  have  already  seen  that  the  oxyhaemo- 
globin  is  only  a  loose  compound,  and  in  the  tissues  it  parts  with 
its  oxygen.  The  oxygen  does  not  necessarily  undergo  immediate 
union  with  carbon  to  form  carbonic  acid,  and  with  hydrogen  to  form 
water,  but  in  most  cases,  as  in  muscle,  is  held  in  reserve  by  the  tissue 
itself.  Ultimately,  however,  these  substances  pass  into  the  venous 
blood,  and  the  carbonic  acid  and  a  portion  of  the  water  find  an  outlet 
by  the  lungs. 

Inspired  and  Expired  Air. — The  composition  of  the  inspired  and 
expired  air  may  be  compared  in  the  following  table  : — 


- 

Inspired  or  atmospheric  Air 

Expired  Air 

16-03  vols,  per  cent. 
79 
4-4       „ 

saturated 
that  of  body  (37°  C.)      ' 

Oxygen      . 

Nitrogen    . 
Carbonic  acid     . 
"Watery  vapour  . 
Temperature 

20-96  vols,  per  cent. 
79 
0-04     „ 

variable 

'  There  may  be  a  slight  reaction  with  the  blood  of  allied  animals  ;  for  instance 
with  monkey's  blood  in  the  case  of  man. 


RESPIEATION  127 

The  nitrogen  remains  unchanged.  The  recently  discovered  gases, 
argon,  crypton,  &c.,  are  in  the  above  table  reckoned  in  with  the 
nitrogen.  They  are,  however,  only  present  in  minute  quantities. 
The  chief  change  is  in  the  proportion  of  oxygen  and  carbonic  acid. 
The  loss  of  oxygen  is  about  5,  the  gain  in  carbonic  acid  4^.  If  the 
inspired  and  expired  airs  are  carefully  measured  at  the  same  tempera- 
ture and  barometric  pressure,  the  volume  of  expired  air  is  thus  rather 
less  than  that  of  the  inspired.  The  conversion  of  oxygen  into  carbonic 
acid  would  not  cause  any  change  in  the  volume  of  the  gas,  for  a 
molecule  of  oxygen  (0^)  would  give  rise  to  a  molecule  of  carbonic  acid 
(CO2)  which  would  occupy  the  same  volume  (Avogadro's  law).  It 
must,  however,  be  remembered  that  carbon  is  not  the  only  element 
which  is  oxidised.  Fats  contain  a  number  of  atoms  of  hydrogen 
which  during  metabolism  are  oxidised  to  form  water ;  a  certain  small 
amount  of  oxygen  is  also  used  in  the  formation  of  urea.  Carbo- 
hydrates contain  sufficient  oxygen  in  their  own  molecules  to  oxidise 
their  hydrogen ;  hence  the  apparent  loss  of  oxygen  is  least  when  a 
vegetable  diet  (that  is,  one  consisting  largely  of  starch  and  other 
carbohydrates)  is  taken,  and  greatest  when  much  fat  and  protein  are 

eaten.     The  quotient  ^—v^-^T^-,     is  called  the  respiratory  quotient. 
O2  absorbed 

Normally  it  is         =0*9,  but  this  varies  considerably  with  diet,  as 
o 

just  stated. 

Gases  of  the  Blood. — From  100  volumes  of  blood  about  60  volumes 
of  gas  can  be  removed  by  the  mercurial  air-pump  (see  Appendix). 
The  average  composition  of  this  gas  from  dog's  blood  is — 


- 

Arterial  Blood 

20 

lto2 

40 

Venous  Blood 

Oxygen 
Nitrogen    . 
Carbonic  acid     . 

8  to  12 

lto2 

46 

The  nitrogen  in  the  blood  is  simply  dissolved  from  the  air  just 
as  water  would  dissolve  it :  it  has  no  physiological  importance.  The 
other  two  gases  are  present  in  much  greater  amount  than  can  be 
explained  by  simple  solution  ;  they  are,  in  fact,  chiefly  present  in 
loose  chemical  combinations.  Less  than  one  volume  of  the  oxygen 
and  about  two  of  carbonic  acid  are  present  in  simple  solution  in  the 
plasma. 

Oxygen  in  the  Blood. — The  amount  of  gas  dissolved  in  a  liquid 
varies  with  the  pressure  of  the  gas ;    double  the  pressure  and  the 


128  ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 

amount  of  gas  dissolved  is  doubled.  The  oxygen  in  the  blood  does 
not  vary  directly  with  oxygen  pressure,  for  the  amount  of  that  gas 
in  simple  solution  forms  only  a  small  fraction  of  the  total  present. 
This  small  amount  is  of  coarse  doubled  by  doubling  the  pressure,  but 
such  an  increase  is  insignificant,  the  bulk  of  the  gas  being  in  chemical 
union  w^ith  haemoglobin.  The  oxygen  of  oxyhtemoglobin  can  be 
replaced  by  equivalent  quantities  of  other  gases,  such  as  carbonic 
oxide.  The  tension  or  partial  pressure  of  oxygen  in  the  air  of  the 
alveoli  is  less  than  that  in  the  atmosphere,  but  greater  than  that  in 
venous  blood;  hence  oxygen  passes  from  the  alveolar  air  into  the 
blood ;  the  oxygen  immediately  combines  with  the  haemoglobin,  and 
thus  leaves  the  plasma  free  to  absorb  more  oxygen  ;  and  this  goes  on 
until  the  haemoglobin  is  entirely,  or  almost  entirely,  saturated  with 
oxygen.  The  reverse  change  occurs  in  the  tissues  where  the  partial 
pressure  of  oxygen  is  lower  than  in  the  plasma,  or  the  lymph  that 
bathes  the  tissue  elements ;  the  plasma  parts  with  its  oxygen  to  the 
lymph,  the  lymph  to  the  tissues  ;  the  oxyhaemoglobin  then  undergoes 
dissociation  to  supply  more  oxygen  to  the  plasma  and  lymph,  and  this 
in  turn  to  the  tissues  once  more.  This  goes  on  until  the  oxyhaemo- 
globin loses  a  great  portion  of  its  store  of  oxygen,  but  even  in  asphyxia 
it  does  not  lose  all. 

The  following  values  are  given  for  the  tension  of  oxygen  in  per- 
centages of  an  atmosphere  : — 

External  air 20-96 

Alveolar  air 13-16     ^ 

Arterial  blood  .....  14  | 

Tissues    . 0 

The  arrow  shows  the  direction  in  which  the  gas  passes. 

The  methods  of  obtaining  the  gases  of  the  blood  and  analysing 
them  are  described  in  the  Appendix.  When  the  gases  are  being 
pumped  off  from  the  blood,  very  little  oxygen  comes  off  until  the 
pressure  is  greatly  reduced,  and  then,  at  a  certain  point,  it  is  suddenly 
disengaged.  This  shows  it  is  not  in  simple  solution,  but  is  united 
chemically  to  the  haemoglobin  as  oxyhaemoglobin,  which  is  dissociated 
when  the  pressure  is  extremely  low. 

The  ability  of  the  tissues  to  form  reduction  products  is  shown  by 
Ehrlich's  experiments  with  methylene  blue  and  similar  pigments. 
Methylene  blue  is  more  stable  than  oxyhaemoglobin  ;  but  if  it  is  injected 
into  the  circulation  of  a  living  animal,  and  the  animal  killed  a  few 
minutes  later,  the  blood  is  found  dark  blue,  but  the  organs  colourless. 
On  exposure  to  oxygen  the  organs  become  blue.  In  other  words 
the   tissues  have  formed  a   colourless   reduction  product  from   the 


RESPIRATION  129 

methylene   blue  ;  on  exposure  to  the   air  this  is  oxidised,  and  the 
blue  pigment  is  thus  regenerated. 

Carbonic  Acid  in  the  Blood. — What  has  been  said  for  oxygen  holds 
good  in  the  reverse  direction  for  carbonic  acid.  Compounds  are 
formed  in  the  tissues  where  the  tension  of  the  gas  is  high  :  these  pass 
into  the  lymph,  then  into  the  blood,  and  in  the  lungs  the  compounds 
undergo  dissociation,  carbonic  acid  passing  into  the  alveolar  air  where 
the  tension  of  the  gas  is  comparatively  low,  though  it  is  greater  here 
than  in  the  expired  air. 

The  relations  of  this  gas  and  the  compounds  it  forms  are  more 
complex  than  in  the  case  of  oxygen.  If  blood  is  divided  into  plasma 
and  corpuscles  it  will  be  found  that  both  yield  carbonic  acid,  but  the 
yield  from  the  plasma  is  the  greater.  If  we  place  blood  in  a  vacuum 
it  bubbles,  and  gives  out  all  its  gases ;  addition  of  a  weak  acid 
causes  no  further  liberation  of  carbonic  acid.  If  plasma  or  serum  is 
similarly  treated  the  gas  comes  off,  but  from  10  to  18  per  cent,  of  the 
carbonic  acid  is  fixed — that  is,  the  addition  of  some  stronger  acid, 
such  as  phosphoric  acid,  is  necessary  to  displace  it.  Fresh  red  cor- 
puscles will,  however,  take  the  place  of  the  phosphoric  acid,  and  thus 
it  has  been  surmised  that  oxyhaemoglobin  has  the  properties  of  an 
acid. 

One  hundred  volumes  of  venous  blood  contain  forty- six  volumes 
of  carbonic  acid.  Whether  this  is  in  solution  or  in  chemical  com- 
bination is  determined  by  ascertaining  the  tension  of  the  gas  in  the 
blood.  One  hundred  volumes  of  blood  plasma  would  ^  dissolve  more 
than  an  equal  volume  of  the  gas  at  atmospheric  pressure[if  its  solu- 
bility in  plasma  were  equal  to  that  in  water.  ^  If,  then,  the 'carbonic 
acid  were  in  a  state  of  solution,  its  tension  would  be  very  high,  but 
it  proves  to  be  only  equal  to  about  5  per  cent,  of  an  atmosphere. 
This  means  that  when  venous  blood  is  brought  into  an  atmosphere 
containing  5  per  cent,  of  carbonic  acid,  the  blood  neither  gives  off 
any  carbonic  acid  nor  takes  up  any.  Hence  the  remainder  of  the 
gas,  95  per  cent.,  is  in  a  condition  of  chemical  combination.  The 
chief  compound  appears  to  be  sodium  bicarbonate. 

The  carbonic  acid  and  phosphoric  acid  of  the  blood  are  in  a  state 
of  constant  struggle  for  the  possession  of  the  sodium.  The  salts 
formed  by  these  two  acids  depend  on  their  relative  masses.  If 
carbonic  acid  is  in  excess  we  get  sodium  carbonate  (NagCOa),  and 
mono-sodium  phosphate  (NaH2P04)  ;  but  if  the  carbonic  acid  is 
diminished,  the  phosphoric  acid  obtains  the  greater  share  of  sodium 

'  To  be  exact,  the  solubility  of  carbon  dioxide  in  plasma  is  a  little  less  than 
in  pure  water. 


130  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

to  form  disodium  phosphate  (Nti2HP04).  In  this  way,  as  soon  as 
the  amount  of  free  carbonic  acid  diminishes,  as  in  the  lungs,  the 
amount  of  carbonic  acid  in  combination  also  decreases ;  whereas  in 
the  tissues,  where  the  tension  of  the  gas  is  highest,  a  large  amount  is 
taken  up  into  the  blood,  where  it  forms  sodium  bicarbonate. 

The  tension  of  the  carbonic  acid  in  the  tissues  is  high,  but  one 
cannot  give  exact  figures ;  we  can  measure  the  tension  of  the  gas  in 
certain  secretions  ;  in  the  urine  it  is  9,  in  the  bile  7  per  cent.  The 
tension  in  the  cells  themselves  must  be  higher  still. 

The  following  figures  (from  Fredericq)  give  the  tension  of  carbonic 
dioxide  in  percentages  of  an  atmosphere  : — 


Tissues    . 
Venous  blood 
Alveolar  air 
External  air 


5  to  9       ) 
3-8  to  5-4    in  dog 
2-8  j 

0-04 


The  arrow  indicates  the  direction  in  which  the  gas  passes  :  namely, 
in  the  direction  of  pressure  from  the  tissues  to  the  atmosphere. 

In  some  other  experiments,  also  on  dogs,  the  following  are  the 

figures  given  : — 

Arterial  blood  .         .         .         .         .  .  2 '8 

Venous  blood    .         .         ".         .         .  .  5*4        ^ 

Alveolar  air      .....  .  3"56       | 

Expired  air       .         .         .         .         .  .  2*8 

It  will  be  seen  from  these  figures  that  the  tension  of  carbonic  acid 
in  the  venous  blood  (5-4)  is  higher  than  in  the  alveolar  air  (3-56) ;  its 
passage  into  the  alveolar  air  is  therefore  intelligible  by  the  laws  of 
diffusion.  Diffusion,  however,  should  cease  when  the  tension  of  the 
gas  in  the  blood  and  in  the  alveolar  air  are  equal.  But  the  transference 
goes  beyond  the  establishment  of  such  an  equilibrium,  for  the  tension 
of  the  gas  in  the  blood  continues  to  sink  until  it  is,  when  the  blood  is 
arterial,  ultimately  less  (2'8)  than  in  the  alveolar  air. 

The  whole  question  is  beset  with  great  difficulties  and  contradic- 
tions. Analyses  by  different  observers  have  given  very  different 
results,  but  if  such  figures  as  those  just  quoted  are  ultimately  found 
to  be  correct,  we  can  only  explain  tliis  apparent  reversal  of  a  law 
of  nature  by  supposing  with  Bohr,  that  the  alveolar  epithelium 
possesses  the  power  of  excreting  carbonic  acid,  just  as  the  cells  of 
secreting  glands  are  able  to  select  certain  materials  from  the  blood 
and  reject  others.  Eecent  work  by  Bohr  and  Haldane  has  also 
shown  that  in  all  probability  the  same  explanation  —  epithelial 
activity — must  be  called  in  to  account  for  the  absorption  of  oxygen. 
Haldane,  in  fact,  states  that  the  tension  of  oxygen  in  the  blood  is 


EESPIRATION  131 

greater  than  iu  the  atraosphere.  In  the  swim-bladder  of  fishes 
(which  is  analogous  to  the  lung  of  mammals)  the  oxygen  is  certainly 
far  in  excess  of  anything  that  can  be  explained  by  mere  diffusion. 
The  storage  of  oxygen,  moreover,  ceases  when  the  vagus  nerves 
which  supply  the  swim-bladder  are  divided. 

Some  Continental  observers  have  stated  that  certain  noxious 
substances  are  ordinarily  contained  in  expired  air,  which  are  much 
more  poisonous  than  carbonic  acid,  but  researches  in  this  country 
have  entirely  failed  to  substantiate  this.  If  precautions  be  taken  by 
absolute  cleanliness  to  prevent  admixture  of  the  air  with  exhalations 
from  skin,  teeth,  and  clothes,  the  expired  air  only  contains  one  noxious 
substance,  and  that  is  carbonic  acid. 

Tension  of  Gases  in  Fluids. — It  is  necessary  to  understand 
thoroughly  the  expression  *  tension  of  gases  in  fluids '  ;  we  will 
therefore  go  into  the  matter  a  little  more  fully. 

The  first  question  which  arises  is,  In  what  circumstances  will  a 
gas,  dissolved  in  a  fluid,  diffuse  out  of  the  fluid  into  the  air  in  contact 
with  it  ?  or  vice  versa,  in  what  circumstances  will  a  gas  diffuse  out  of 
the  air  into  the  fluid,  and  at  what  rate  will  it  do  so  ? 

The  answer  depends  upon  the  physical  constants  of  the  fluid  and 
of  the  atmosphere ;  and  these  must  be  determined  experimentally. 

As  an  example  the  following  instance  may  be  taken : — 100  c.c. 
of  water  charged  with  80  c.c.  of  carbonic  acid  are  shaken  with  pure 
air  in  a  closed  bottle  of  500  c.c.  capacity.  The  carbonic  acid  will 
come  out  of  the  water  at  first,  but  as  the  shaking  continues  the 
carbonic  acid  will  come  out  more  and  more  slowly  until  it  entirely 
ceases  to  do  so.  Analysis  of  the  air  and  of  the  water  in  the  bottle 
would  show  that  the  water  had  not  parted  with  all  its  carbonic  acid. 
It  would  be  found  that  the  water  contained  16  c.c.  of  carbonic  acid 
dissolved  in  it,  while  64  c.c.  have  diffused  out  into  the  air. 

At  the  end  of  this  experiment,  then,  there  will  be  in  the  atmosphere 
64  c.c.  of  carbonic  acid  in  a  space  of  400  c.c.  If  the  whole  space 
(400  c.c.)  were  filled  with  carbonic  acid  its  pressure  would  be  760  mm. 
The  partial  pressure  of  carbonic  acid  in  the  atmosphere  of  the  bottle 
is  therefore  760  x  /q^o  mm-  of  mercury =122  mm.  Water,  therefore, 
containing  16  volumes  per  cent,  of  carbonic  acid  is  in  equilibrium 
with  an  atmosphere  in  which  the  carbonic  acid  exerts  a  pressure  of 
122  mm.  This  fact  is  stated  shortly  by  the  phrase  *  when  water  has 
a  carbonic  acid  tension  of  122  mm.  it  contains  16  per  cent,  of 
carbonic  acid.' 

The  amount  of  carbonic  acid  which  would  be  contained  in  any 
other  fluid  when  it  was  in  equilibrium  with  an  atmosphere  having  a 

k2 


132 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


122  mm.  pressure  of  carbonic  acid  would  be  different  from  that 
contained  in  water.  For  instance,  alcohol  would  contain  50  per  cent, 
of  the  gas.  In  both  fluids  the  tension  of  carbonic  acid  is  the  same 
but  the  quantity  of  carbonic  acid  would  be  as  stated,  and  therefore 
different. 

The  tension  of  a  gas  in  a  liquid,  therefore,  is  the  pressure  of  that 
gas  in  an  atmosphere  of  such  a  composition,  that  the  liquid  would 
neither  acquire  that  gas  from  the  atmosphere  nor  impart  that  gas  to 
it,  if  the  liquid  and  the  atmosphere  were  brought  into  intimate  contact, 
as  by  shaking. 

Measurement  of  Tension  in  Fluids — Aerotonometer.— The  measure- 
ment of  the  tension  of  gases  in  fluids  is  conducted  upon  the  principles 
of  the  example  given  above.  The  instrument  for 
the  purpose  is  called  an  aerotonometer.  The  form 
used  by  Loewy  consists  simply  of  a  closed  bottle, 
into  which  the  blood  and  the  air  can  be  put,  and  from 
which  they  can  be  withdrawn  by  suitable  means. 
Through  the  stopper  of  the  bottle  three  tubes  pass — 
A,  B,  and  c— each  of  which  is  provided  with  a  piece 
of  rubber  tubing  and  a  clip.  The  tube  A  is  used 
for  introducing  or  expelling  the  blood  (e).  c  is 
used  for  introducing  or  expelling  the  air ;  b  is  con- 
nected with  a  rubber  bag  containing  water  inside 
Loewy's  Agrotouometer.  the  bottlc,  while  outside  a  connection  can  be  made 
with  a  syringe.  A  little  mercury  should  be  put  into 
the  bottle  to  defibrinate  the  blood.  To  determine  the  carbonic  acid 
tension  in  blood  several  bottles  should  be  filled  with  gases  of  known 
composition  from  gasometers  before  the  experiment.  Into  each  bottle 
some  blood  is  drawn  from  the  animal.  This  is  done  by  attaching  a  to 
a  cannula  in  one  of  its  vessels,  and  then,  when  water  is  withdrawn 
from  the  bag  d  by  the  syringe,  a  corresponding  amount  of  blood  enters 
the  aerotonometer.  Each  bottle  is  shaken  violently  for  some  time. 
When  equilibrium  has  been  estabhshed  the  air  can  be  taken  from 
the  air  space  f  by  attaching  c  to  an  air-analysis  apparatus,  and  forcing 
water  into  the  bag  d  from  the  syringe  attached  to  b.  An  example  may 
illustrate  the  result  which  might  be  obtained. 

Determination  of  carbonic  acid  tenaion  of  blood. 

Bottle  .... 

Initial  percentage  of  the  gas 
Final  percentage  of  gas  present 

From  the  above  figures  it  will  be  seen  that  the  blood  has  acquired 


Fig.  38. 


I 

II 

III 

IV 

V 

6-0 

5-5 

5-0 

4-5 

4 

5-8 

5-4 

5-1 

4-7 

4-8 

RESPIRATION 


133 


carbonic  acid  from  the  air  in  bottles  I  and  II,  while  the  reverse  has 
taken  place  in  bottles  III,  IV,  and  V.  The  carbonic  acid  tension  of 
the  blood  is  therefore  between  5'4:  and  5*1  per  cent. 

Relation  of  Tension  to  Composition. — The  aerotonometer  may  be 
used  to  determine  the  relation  of  the  tension  of  a  gas  in  the  blood 
(oxygen  or  carbonic  acid)  to  the  quantity  contained  in  that  fluid. 
The  example  given  above  might  be  extended  for  this  purpose  in  the 
following  way.  After  the  air  has  been  w^ithdrawn  for  analysis,  it  is 
only  necessary  to  attach  a  to  the  blood-bulb  of  a  mercurial  gas  pump, 
and,  by  forcing  more  water  into  b,  expel  a  sufficient  quantity  of 
blood  for  analysis.  If  an  analysis  was  made  for  each  of  the  bottles, 
the  quantity  of  carbonic  acid  would  be  determined  corresponding  to 
the  tensions,  5'8,  5"4,  5*1,  4'7,  and  4-3  per  cent,  respectively. 

The  comparision  of  the  quantity  of  carbonic  acid  in  the  blood 
with  its  tension  is  not  of  importance,  but  the  corresponding  measure- 
ments for  oxygen  have  been  carried  out  with  great  care  by  a  number 
of  observers,  both  for  blood  and  for  solutions  of  haemoglobin. 

It  is  found  that  the  two  sets  of  observations  agree  very  closely, 
and  this  fact  forms  the  most  substantial  evidence  for  our  belief  that 
the  oxygen  in  the  blood  is  almost  entirely  associated  with  the  haemo- 
globin. 

The  relation  of  the  quantity  of  oxygen  in  the  blood  to  the  tension 
which  it  exerts  may  be  conveniently  set  forth  in  a  curve,  which  is 
called  the  dissociation  curve  of  oxyhaemoglobin. 

An  example  may  illustrate 
the  use  to  which  the  informa- 
tion, given  in  this  curve,  may 
be  put.  The  amount  of  blood 
which  comes,  say,  from  the 
pancreas  is  too  small  to  allow^ 
of  a  direct  determination  of  the 
tension  of  the  oxygen  which  it 
contains.  We  can,  however, 
calculate  it  from  the  following 
data.  (1)  The  amount  of  oxygen 
which  the  blood  would  contain 
if  saturated  (this  can  be  arrived 

at  by  saturating  arterial  blood  and  analysing  it).     (2)  The  amount  of 
"oxygen  in  the  venous  blood.     In  an  actual  experiment  (1 )  was  22*0  c.c, 


^ 

^-^ — r"t"1  i  ! 

/ 

H^ 

^-" 

U   M   M 

/  . 

/" 

/ 

/ 

'\f> 

\\\ 

,.l 

/■ 

"\l 

ll- 

_.JJ 

_i 

n^  20   to    43   so   £}   70   tO   DO   100  ItJ   120  133  MO 

i;.  3!).— Dissociation  of  oxygen  curves  for  Wood  (n) 
and  Li«;moglobin  solution  (h)  at  38°  C.  The 
figures  along  the  base  line  are  pressures  in  milli- 
metres of  mercury,  aud  those  along  the  ordinate 
are  percentages  of  oxygen.     (Bohr.) 


(2)  was  10-7  c.c 

By  referring   to   the   dissociation 


10-7 


The  saturation  then  was  100  x  ^^  =  49 


per   cent, 
curve   w^e   see  that  49  per  cent. 


184  ESSENTIALS  OF  CHEMICAL  rHYSTOLOGY 

saturation  corresponds  with  an  oxygen  tension  of  about  14  mm.  of 
mercm'y,  or  1*8  per  cent,  of  an  atmosphere. 

The  Carbonic  Oxide  Method  of  Estimating  the  Oxygen  Tension  of  Arterial 
Blood. — This  method  was  devised  by  Haldane,  and  is  considered  by  him  and 
Lorrain  Smith  to  give  more  trustworthy  results  than  those  obtained  by  the 
aerotonometer.  If  blood  is  exposed  to  a  mixture  of  carbonic  oxide  and 
oxygen,  the  haemoglobin  will  become  saturated  by  these  gases  according  to 
their  relative  tensions.  If  a  number  of  experiments  are  performed  using 
different  percentages  of  carbonic  oxide,  the  results  may  be  expressed 
graphically  as  the  curve  of  dissociation  of  carboxyhaemoglobin  in  air.  When, 
in  place  of  such  experiments  in  vitro,  an  animal  is  made  to  breathe  air  contain- 
ing a  known  percentage  of  carbonic  oxide,  the  comparison  of  the  saturation 
of  its  blood  with  the  saturation  of  its  blood  in  vitro,  exposed  to  the  same 
percentage  of  carbonic  oxide  in  air  (which  has  an  oxygen  tension  of  20*9  per 
cent.)  gives  us  the  means  of  discovering  the  oxygen  tension  in  the  arterial 
blood  of  the  lung  capillaries  :  this  will  be  higher  or  lower  than  that  of  the  air 
according  as  the  saturation  by  carbonic  oxide  is  correspondingly  lower  or  higher. 
A  small  animal  like  a  mouse  is  made  to  breathe  air  containing  a  known  per- 
centage of  carbonic  oxide.  After  a  sufficient  time  the  animal  is  killed  and 
the  amount  of  carboxyhaemoglobin  is  determined  colorimetrically  in  a  drop 
of  its  blood.  The  data  thus  obtained  are  compared  with  the  data  previously 
expressed  in  the  curve  of  dissociation  of  carboxyhaemoglobin  in  air  ;  it  is 
then  easy  to  calculate  whether  the  oxygen  tension  in  the  blood  is  higher  or 
lower  than  that  of  air.  The  results  of  the  method  show  generally  that  the 
tension  of  oxygen  in  the  arterial  blood  as  it  leaves  the  lungs  is  liigher  than 
could  result  from  simple  diffusion  of  the  oxygen  through  the  alveolar 
epithelium  ;  in  other  words,  the  epithelial  cells  are  capable  of  secreting  oxygen 
into  the  blood  until  an  oxygen  pressure  is  reached  considerably  above  that  in 
the  alveolar  air. 

The  results  expressed  in  percentages  of  an  atmosphere  are  as  follows  : — 
Oxygen  tension  of  arterial  blood  in  man,  38-5;  in  mouse,  22*6;  in  dog. 
21 ;  in  cat,  35-3  ;  in  rabbit,  27*6,  and  in  birds  30  to  50  per  cent.  The  results 
in  the  case  of  man  and  larger  animals  probably  require  revision,  as  it  is  not 
certain  that  the  time  allowed  for  the  establishment  of  the  balance  of  carbonic 
oxide  and  oxygen  has  been  sufficient  in  any  of  the  experiments. 

Tissue-Respiration. — Before  the  processes  of  respiration  were  fully 
understood  the  lungs  were  looked  upon  as  the  seat  of  combustion  ; 
they  were  regarded  as  the  stove  for  the  rest  of  the  body  where  effete 
material  was  brought  by  the  venous  blood  to  be  burnt  up.  When  it 
was  shown  that  the  venous  blood  going  to  the  lungs  already  contained 
carbonic  acid,  and  that  the  temperature  of  the  lungs  is  not  greater 
than  that  of  the  rest  of  the  body,  this  explanation  had  of  necessity  to 
be  dropped. 

Physiologists  next  transferred  the  seat  of  the  combustion  to  the 
blood ;  but  since  then  innumerable  facts  and  experiments  have 
shown  that  it  is  in  the  tissues  themselves,  and  not  in  the  blood,  that 
combustion  occurs.  The  methylene-blue  experiment  already  described 
(p.  128)  shows  this ;  and  the  following  experiment  is  also  quite  con- 
clusive.    A  frog  can  be  kept  alive  for  some  time  after  salt  solution  is 


IlESPI  RATIO  X 


135 


is  necessary  (1)  to 
analyse   the   blood 


#0^ 


substituted  for  its  blood.  The  metabolism  goes  on  actively  if  the 
animal  is  kept  in  pure  oxygen.  The  taking  up  of  oxygen  and  giving 
out  of  carbonic  acid  must  therefore  occur  in  the  tissues,  as  the  animal 
has  no  blood. 

The  following  are  the  amounts  of  oxygen  used  up  per  minute  by 
one  gramme  of  certain  epithelial  and  muscular  organs  respectively  : — 
Submaxillary  gland  004  c.c,  pancreas  0-05  c.c,  kidney  O'OS  c.c, 
heart  (contracting  very  feebly  and  slowly)  0-007  c.c,  muscles  of  leg 
(with  spinal  cord  destroyed)  0*003  c.c. 

In  order  to  obtain  data,  such  as  the  above,  it 
analyse  the  blood  going  to  the  organ ;  (2)  to 
emerging  from  the  organ ;  (3)  to  determine  the 
amount  of  blood  passing  through  the  organ  in 
one  minute. 

Analysis  of  the  blood  may  be  performed 
by  either  of  two  methods,  the  mercurial  air- 
pump  (see  Appendix)  or  the  chemical  method  of 
expelling  the  oxygen  and  carbonic  acid  from  the 
blood  by  means  of  potassium  ferricyanide  and 
phosphoric  acid  respectively. 

Chemical  Method  of  Blood  Gas  Analysis. — 
When  a  solution  of  hsemoglobin  is  treated  W'ith 
potassium  ferricyanide  it  yields  all  its  oxygen  to 
the  air  on  shaking,  just  as  urea  yields  its  nitrogen 
when  treated  with  sodium  hypobromite.  The 
apparatus  used  for  determining  the  oxygen  in 
blood  is  very  similar  to  a  Dupre's  urea  apparatus 
(see  p.  142).  The  blood  (5  c.c.)  is  placed  in  the 
large  bottle  (f)  (fig.  40)  underneath  a  layer  of 
dilute  ammonia  solution.  The  blood  is  thus  pro- 
tected from  the  air,  while  the  apparatus  becomes 
equal  in  temperature  to  the  bath  in  which  it  is 
placed.  The  blood  is  shaken  with  the  ammonia 
solution  which  lakes  it  thoroughly;  the  ferri- 
cyanide solution  is  then  spilt  into  the  laked  blood 
from  the  tube  g,  and  the  oxj^gen  is  shaken  out  of  the  solution. 
When  the  oxygen  has  been  determined  the  bottle  is  opened  and 
phosphoric  acid  is  placed  in  the  small  tube  g  :  this  is  subsequently 
spilt  into  the  mixture  of  blood,  ammonia,  and  ferricyanide  ;  it  liberates 
the  carbonic  acid,  w^hich  is  also  shaken  out  of  the  fluid.  The  carbonic 
acid  does  not  come  completely  out,  however,  and  a  con-ection  has  to  be 
introduced  for  the  quantity  w^hich  remains  in  solution.    The  gas  w^hich 


Fio.  40.— Barcroft's  appa- 
ratus for  obtaining  tJie 
blood  gases. 


136 


ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 


comes  off  passes  into  the  tube  c,  which  was  originally  filled  up  to  the 
zero  mark  with  water  in  continuity  with  that  in  the  reservoir  a.  This 
would  depress  the  column  of  fluid  in  c  and  raise  that  in  the  open 
tube  D,  which  is  graduated  in  millimetres.  In  practice,  however,  it 
is  convenient  to  keep  the  gas  always  at  the  same  volume  :  this  may 
be  done  by  raising  the  pressure  in  the  open  tube  (d)  by  squeezing 
some  of  the  water  with  which  the  gauge  is  filled  out  of  a  rubber 
reservoir  b,  which  forms  the  base  of  the  gauge  ;  thus  the  level  of  the 
water  in  c  is  kept  at  the  zero,  while  that  in  d  rises  from  h  to  i. 
The  actual  measurement  then  is  the  increase  of  pressure  {i.e.  the 
height  of  the  column  of  water  hi)  which  is  necessary  to  keep  the 
gases  in  the  tube  c  at  the  same  volume  as  that  which  was  previously 
occupied  by  the  air  in  that  tube.  From  this  the  quantity  coming  off 
can  be  calculated. 

The  chemical  method  is  not  quite  so  accurate  as  the  vacuum  pump, 
but  it  is  much  more  convenient  for  the  study  of  many  problems,  as  it 
requires  less  blood,  and,  owing  to  its  simplicity,  a  great  number  of 
observations  can  be  made  upon  a  single  animal. 

Changes  in  Tissue  Respiration  caused  by  Activity.— In  all  organs, 
so  far  as  is  known,  increased  activity  is  associated  with  increased 
tissue  respiration.     The  increase  is  commonly  three-  to  six-fold.     It  is 


Organ 

1                  1 

'■      Oxygen      i 
Condition  of  rest           used  per         Condition  of  activity 
1       minute      , 

Oxygen  used 
per  minute 

Muscle  . 

Nerves  cut 

1 
1 

0-003  c.c.  i     Tonic  (nerves 

nncut) 

0-006  c.c. 
0-020  c.c. 

Heart    . 

Very  slow  and 
feeble  contrac- 
tions 

0-007  c.c. !  Normal  contrac- 
tions 
Very  active 

1 

0-014 
0-054 

Submaxillary 
gland 

Nerves  cut       '  0*04  c.c.    '  Chorda  tympani 

stimulated 

1 

0-12  c.c. 
0-20            1 

1 

Pancreas 

i 

Not  secreting 

0-05  c.c.     Secretion  after  in- 
jection of  secre- 
tin 

1 
Kidney . 

Scanty 
secretion 

0-03  c.c.     After  injection  of 
a  diuretic 

i 

007  c.c. 

KESPIRATION 


137 


often  more  easy  to  demonstrate  the  augmented  oxygen  consumption 
than  the  augmented  output  of  carbonic  acid.  This  is  due  to  several 
causes  :  (1)  carbonic  acid  is  soluble  in  the  tissues  in  which  it  is  pro- 
duced; (2)  any  change  in  the  chemical  reaction  of  the  tissues  alters 
the  amount  of  carbonic  acid  which  they  give  out  to  the  blood  ;  if,  for 
instance,  it  becomes  more  alkaline  it  retains  a  portion  of  its  carbonic 
acid. 

The  preceding  table  shows  the  variations  which  take  place  in  the 
oxygen  intake  of  several  organs  as  the  result  of  activity  produced  by 
widely  different  forms  of  stimulus  (Barcroft). 

The  relation  of  the  oxygen  taken  in  to  the  carbonic  acid  given  out 
is  well  shown  in  the  following  experiment  performed  by  Zuntz  on  the 
leg  of  the  dog  : — 


Blood-vessel 

Gases  in  blood  per  cent. 

Remarks 

Femoral  vein 
1  Carotid  artery 

1 

Oxygeu 

1-2 
14-4 

(Jarbon 
dioxide 

36-32 
4-92 

Muscles  tonic,  nerves 
being  intact 

1 

j  Exchange 

i 

1  Femoral  vein 
Carotid  artery 

18-20 

14-4 

2-85 
13-30 

33-16 
23-06 

10-1 

After  section  of  sciatic 
and  crural 

Exchange 

10-45 

Two  points  must  be  noted  in  considering  the  above  table  : — 

(1)  That  the  exchange  of  gases  was  decreased  on  cutting  the 
nerves.  The  decrease  in  metabolism  was  greater  even  than  the 
figures  show,  for  the  blood  flow  through  the  leg  was  decreased. 

(2)  That  the  oxygen  exchange  and  the  carbonic  acid  exchange 

changed  in  about  the  same  proportions.     The  ratio  of  the  carbonic 

14-4 
acid  given  out  to  the  oxygen  taken  in  was  -^-^  with  the  nerves  intact 

±o'2 


and 


lO'l 
10-45 


with  the  nerves  cut. 


Effect  of  reduced  Oxygen  Tension  on  Tissue  Respiration. — When 
the  oxygen  tension  in  the  blood  is  reduced,  the  tissues  still  take  up  the 
same  quantity  of  oxygen  as  before  and  give  out  as  much,  or  slightly 


138  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

more,  carbonic  acid ;  thus,  in  the  case  of  a  dog,  when  the  oxygen 
tension  in  the  blood  is  approximately  18  millimetres  of  mercury  or 
one  fortieth  of  an  atmosphere,  163  c.c.  of  oxygen  per  minute  were 
used  up  by  the  animal,  and  172  c.c.  of  carbonic  acid  were  given  out ; 
normally  by  the  same  animal  the  oxj^gen  consumption  was  157  c.c. 
and  the  carbonic  acid  output  158  c.c.  The  reason  why  the  tissues 
extract  oxygen  with  such  readiness  from  the  blood,  even  when  the 
oxygen  exists  in  the  blood  at  a  low  tension,  is  that  there  is  no  free 
oxygen  in  the  tissues  themselves  (and  they  always  thirst  for  it).  This 
fact  can  be  demonstrated  in  more  than  one  way. 

(1)  No  oxygen  can  be  extracted  from  the  tissues  by  exposing  them 
to  the  vacuum  of  an  air-pump. 

(2)  The  tissues  possess  the  power  of  reducing  such  substances  as 
methylene  blue  (see  p.  128). 

Respiration  in  excised  Tissues.— Excised  frog's  muscle  retains  its 
power  of  contraction  for  a  considerable  time.  During  this  time  it 
gives  out  carbonic  acid.  These  facts  are  true  whether  the  muscle  be 
in  air  or  in  nitrogen.  In  either  case  the  muscle  must  be  regarded  as 
partially  or  entirely  asphyxiated,  for  the  individual  elements  of  the 
muscle  are  cut  off  from  that  ready  supply  of  oxygen  which  normally 
reaches  them.  During  life  (and  the  living  condition  can  be  imitated 
by  placing  an  excised  muscle  in  an  atmosphere  of  pure  oxygen)  the 
muscular  substance  breaks  down  into  a  number  of  somewhat  simpler 
substances  :  one  of  these  is  carbonic  acid.  The  others,  however,  or 
some  of  them,  are  at  once  built  up  again  with  the  inclusion  of  oxygen 
and  some  carbon-containing  substance,  perhaps  sugar,  into  living 
material.  The  muscle,  therefore,  does  not  contain  any  of  the  by- 
products of  its  own  metabolism.  In  excised  muscle,  when  the  oxygen 
supply  is  deficient  the  by-products  accumulate,  as  a  result  of  which 
very  striking  alterations  take  place.  (1)  The  reaction  of  the  muscle 
becomes  acid  and  the  phenomena  of  fatigue  and  functional  death  set 
in.  (2)  The  proteins  become  coagulated,  and  this  is  the  phj^sical  basis 
of  rigor  mortis. 

The  Chemical  Stimulus  to  Respiration. — Haldane  and  Priestley 
have  introduced  a  new  and  simple  method  of  obtaining  the  com- 
position of  the  air  in  the  alveoli.  It  consists  in  collecting  one 
sample  of  air  expelled  by  a  deep  expiration  at  the  end  of  a  quiet 
inspiration,  and  another  of  the  air  expelled  by  a  deep  expiration  at 
the  end  of  a  quiet  expiration  ;  the  mean  of  the  two  gives  the  com- 
position of  alveolar  air.  This  is  much  simpler  than  the  method 
formerly  employed  by  Pfliiger,  which  consisted  in  pumping  off  the 
air  from  an  occluded  portion  of  a  dog's  lung  by  the  means  of  a  fine 


RESPIRATION  189 

elastic  catheter  introduced  via  the  trachea  and  bronchus.  It  has  the 
further  advantage  that  it  can  be  appHed  to  the  human  subject. 

They  found  that  under  constant  atmospheric  pressure  in  man,  the 
alveolar  air  contains  a  nearly  constant  percentage  of  carbon  dioxide  in 
the  same  person.  In  different  individuals  this  percentage  varies 
somewhat,  but  averages  in  men  o'lG,  in  women  and  children  4-77  per 
cent,  of  an  atmosphere.  With  varying  atmospheric  pressures  the 
percentage  varies  inversely  as  the  atmospheric  pressure,  so  that  the 
pressure  or  tension  of  the  carbon  dioxide  remains  constant.  The 
oxygen  pressure,  however,  varies  widely  under  the  same  con- 
ditions. 

These  observations  and  the  next  to  be  immediately  described 
furnish  the  chemical  key  to  the  cause  of  the  amount  of  pulmonary 
ventilation,  and  play  an  important  part  ill  conjunction  with  the 
respiratory  nervous  system  in  the  regulation  of  breathing.  For  the 
respiratory  centre  is  not  only  affected  by  the  impulses  reaching  it  by 
the  vagi  and  other  afferent  nerves,  but  it  is  also  very  sensitive  to  any 
rise  in  the  tension  of  carbon  dioxide  in  the  blood  that  supplies  it. 
The  changes  in  the  tension  of  this  gas  in  the  arterial  blood  are 
normally  proportional  to  the  changes  in  the  carbon-dioxide  pressure 
in  the  alveoli,  and  thus  the  changes  in  the  lung  alveoli  are  trans- 
mitted to  the  respiratory  centre.  They  found  that  a  rise  of  0"2  per 
cent,  in  the  alveolar  carbon-dioxide  pressure  is  sufficient  to  double 
the  amount  of  alveolar  ventilation  during  rest.  During  work  the 
alveolar  carbon-dioxide  pressure  increases  slightly,  and  the  pulmonary 
ventilation  is  consequently  increased.  Changes  in  the  oxygen  pressure 
within  wide  limits  have  no  such  influence ;  the  normal  chemical 
stimulus  to  respiration  is  therefore  the  presence  of  an  increase  of  carbon 
dioxide,  and  not  diminution  of  oxygen.  If  these  limits  are  exceeded, 
as  when  the  oxygen  pressure  falls  below  13  per  cent,  of  an  atmosphere, 
the  respiratory  centre  begins  to  be  excited  by  want  of  oxygen,  for 
the  alveolar  carbon -dioxide  pressure  is  lower  than  normal  under  such 
circumstances.  '  Apnoea  'is  the  name  given  to  the  cessation  of  breathing 
which  temporarily  follows  excessive  ventilation  of  the  lungs,  as  when 
one  takes  a  number  of  deep  breaths  in  rapid  succession.  The  deep 
and  rapid  breathing  clears  out  the  carbon  dioxide  in  the  alveoli  until 
it  is  so  small  in  quantity  that  it  is  insufficient  to  excite  the  respiratory 
centre  via  the  blood  to  action,  the  oxygen  pressure  at  the  same  time 
remaining  sufficiently  high  not  to  excite  the  centre  either.  Hence 
breathing  ceases.  The  old  idea  that  apnoea  is  due  to  over-oxygena- 
tion  of  the  blood  has  been  abundantly  disproved,  but  Head  and  others 
have  gone  to  an  extreme  in  assuming  that  apncea  is  purely  nervous 


140  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

in  origin.  Haldane  considers  it  is  unnecessary  to  assume  the 
existence  of  a  vagus  apncea  in  man  at  all  under  normal  circum- 
stances. It  is,  however,  probable  that  in  normal  breathing  the 
nervous  reflex  and  the  chemical  stimulus  have  both  to  be  reckoned 
with,  though  the  relative  importance  of  these  two  factors  is  a  question 
for  the  future. 


LESSON   X 

URINE 

1.  Test  the  reaction  of  urine  on  litmus  paper. 

2.  Determine  its  specific  gravity  by  the  urinometer. 

3.  Test  for  the  following  inorganic  salts  : 

{a)  Chlorides. — ^ Acidulate  with  nitric  acid  and  add  silver  nitrate;  a  white 
precipitate  of  silver  chloride,  soluble  in  ammonia,  is  produced.  The  object 
of  acidulating  with  nitric  acid  is  to  prevent  phosphates  being  precipitated  by 
the  nitrate  of  silver. 

{h)  Suljjhates. — Acidulate  with  hydrochloric  acid  and  add  barium  chloride. 
x\  white  precipitate  of  barium  sulphate  is  produced.  Hydrochloric  acid  is 
again  added  first,  to  prevent  precipitation  of  phosphates. 

(c)  Phospliates. — i.  Add  ammonia ;  a  white  crystalline  precipitate  of 
earthy  (that  is,  calcium  and  magnesium)  phosphates  is  produced.  This 
becomes  more  apparent  on  standing.  The  alJfaline  (that  is,  sodium  and 
potassium)  phosphates  remain  in  solution. 

ii.  Mix  another  portion  of  urine  with  half  its  volume  of  nitric  acid  ;  add 
ammonium  molybdate,  and  boil.  A  yellow  crystalline  precipitate  falls. 
This  test  is  given  by  both  kinds  of  phosphates. 

4.  Urea. — Take  some  urea  crystals.  Observe  that  they  are  readily  soluble 
in  water,  and  that  effervescence  occurs  when  fuming  nitric  acid  {i.e.  nitric 
acid  containing  nitrous  acid  in  solution)  is  added  to  the  solution.  The 
effervescence  is  due  to  the  breaking  up  of  the  urea.  Carbonic  acid  and 
nitrogen  come  oft'.  A  similar  bubbling,  due  to  evolution  of  nitrogen,  occurs 
when  an  alkaline  solution  of  sodium  hypobromite  is  added  to  another 
portion  of  the  solution. 

5.  Heat  some  urea  crystals  in  a  dry  test-tube.  Biuret  is  formed,  and 
ammonia  comes  off.  Add  a  drop  of  copper-sulphate  solution  and  a  few  drops 
of  20-per-cent.  potash.     A  rose-red  colour  is  produced. 

6.  Quantitative  estimation  of  urea. 

For  this  purpose  Dupre's  apparatus  (fig.  41)  is  the  most  convenient.  It 
consists  of  a  bottle  united  to  a  measuring  tube  by  indiarubber  tubing.  The 
measuring  tube  {an  inverted  burette  will  do  very  well)  is  placed  within  a 
cylinder  of  water,  and  can  be  raised  and  lowered  at  will.  Measure  25  c.c.  of 
alkaline  solution  of  sodium  hypobromite  (made  by  mixing  2  c.c.  of  bromine 
witli  23  c.c.  of  a  40-per-cent.  solution  of  caustic  soda)  into  the  bottle. 
Measure  5  c.c.  of  urine  into  a  small  tube,  and  lower  it  carefully,  so  that  no 
urine  spills,  into  the  bottle.  Close  the  bottle  securely  with  a  stopper  per- 
forated by  a  glass  tube  :  this  glass  tube  ^  is  connected  to  the  measuring  tube  by 

*  The  efficiency  of  the  apparatus  is  increased  by  having  a  glass  bulb  blown  on 
this  tube  to  prevent  froth  passing  into  the  rest  of  the  apparatus.  This  is  not 
shown  in  the  figure. 


142 


ESSENTIALS   OF  CHEMICAL  PHYSIOLOaY 


indiarubber  tubing  and  a  T-piece.  The  third  limb  of  the  T-piece  is  closed 
by  a  piece  of  indiarubber  tubing  and  a  pinch-cock,  seen  at  the  top  of  the 
tigure.     Open  the  pinch-cock  and  lower  the  measuring  tube  until  tlie  surface 

of  the  water  with  which  the  outer  cylin- 
der is  filled  is  at  the  zero  point  of  the 
graduation.  Close  the  pinch- cock,  and 
raise  the  measuring  tube  to  ascertain 
whether  the  apparatus  is  air-tight. 
Then  lower  it  again.  Tilt  the  bottle  so 
as  to  upset  the  urine,  and  shake  well 
for  a  minute  or  so.  During  this  time 
there  is  an  evolution  of  gas.  Then 
immerse  the  bottle  in  a  large  beaker 
containing  water  of  the  same  tempera- 
ture as  that  in  the  cylinder.  After 
two  or  three  minutes  raise  the  measiu-- 
ing  tube  until  the  surfaces  of  the 
water  inside  and  outside  it  are  at  the 
same  level.  Head  off  the  amount  of 
gas  evolved.  This  is  nitrogen.  The 
carbonic  acid  resulting  from  the  de- 
composition of  urea  has  been  absorbed 
by  the  excess  of  soda  in  the  bottle. 
35*4  c.c.  of  nitrogen  are  yielded  by 
0*1  gramme  of  urea.  From  this  the 
(juantity  of  urea  in  the  5  c.c.  of  urine 
and  the  percentage  of  urea  can  be 
calculated.  If  the  total  urea  passed 
in  the  twenty-four  hours  is  to  be 
ascertained,  the  twenty-four  hours' 
urine  must  be  carefully  measured  and 
thoroughly  mixed.  A  sample  is  then 
taken  from  the  total  for  analysis  ;  and 
then,  by  a  simple  'sum  in  proportion, 
the  total  amount  of  urea  is  ascertained. 
7.  Creatinine, — This  substance  may 
be  detected  by  adding  a  little  sodium 
nitro-prusside  and  caustic  soda  to  the 
urine.  A  red  colour  develops,  which 
FIG.  41.-Dnpre"s  urea  apparatus.  ^^^^^  on  boiling. 


The  kidney  is  a  compound  tubular  gland,  the  tubules  of  which  it 
is  composed  differing  much  in  the  character  of  the  epithelium  that 
lines  them  in  various  parts  of  their  course.  The  true  secreting  part 
of  the  kidney  is  the  glandular  epithelium  that  lines  the  convoluted 
portion  of  the  tubules  ;  there  is  in  addition  to  this  what  is  usually 
termed  the  filtering  apparatus  :  tufts  of  capillary  blood-vessels  called 
the  Malpighian  glomeruli  are  supplied  with  afferent  vessels  from  the 
renal  artery;  the  efferent  vessels  that  leave  these  have  a  smaller 
calibre,  and  thus  there  is  high  pressure  in  the  Malpighian  capillaries. 
Certain  constituents  of  the  blood,  especially  water  and  salts,  pass 


URINE  143 

through  the  thin  walls  of  these  vessels  into  the  surrounding  Bow- 
man's capsule,  which  forms  the  commencement  of  each  renal  tubule. 
Bowman's  capsule  is  lined  by  a  flattened  epithelium,  which  is  reflected 
over  the  capillary  tuft.  Though  the  process  which  occurs  here  is 
generally  spoken  of  as  a  filtration,  yet  it  is  no  purely  mechanical 
process,  but  the  cells  undoubtedly  exercise  a  selective  influence,  and, 
among  other  things,  prevent  the  albuminous  constituents  of  the  blood 
from  escaping.  During  the  passage  of  this  dilute  urine  through  the 
rest  of  the  renal  tubule  it  gains  the  constituents,  urea,  urates,  &c., 
which  are  poured  into  it  by  the  secreting  cells  of  the  convoluted 
tubules. 

GENERAL  CHARACTERS  OF  URINE 

Quantity. — A  man  of  average  weight  and  height  passes  from  1,400 
to  1,600  c.c,  or  about  50  oz.,  daily.  This  contains  about  50  grammes 
(1^  oz.)  of  solids.  The  urine  should  be  collected  in  a  tall  graduated 
glass  vessel  capable  of  holding  3,000  c.c,  which  should  have  a 
smooth-edged  neck  accurately  covered  by  a  ground-glass  plate  to 
exclude  dust  and  avoid  evaporation.  From  the  total  quantity  thus 
collected  in  the  twenty-four  hours  samples  should  be  drawn  off  for 
examination. 

Colour. — This  is  some  shade  of  yellow  which  varies  considerably 
in  health  with  the  concentration  of  the  urine.  It  appears  to  be  due 
to  a  mixture  of  pigments  :  of  these  urobilin  is  the  one  of  which  we 
have  the  most  accurate  knowledge.  Urobilin  has  a  reddish  tint  and 
is  ultimately  derived  from  the  blood  pigment,  and,  like  bile  pigment, 
is  an  iron-free  derivative  of  haemoglobin.  The  bile  pigment  (and 
possibly  also  the  hsematin  of  the  food)  is  in  the  intestines  converted 
into  stercobilin  ;  most  of  the  stercobilin  leaves  the  body  with  the 
faeces ;  but  some  is  reabsorbed  and  is  excreted  with  the  urine  as 
urobilin.  Urobilin  is  very  like  the  artificial  reduction  product  of 
bilirubin  called  hydrobilirubin  (see  p.  92).  Normal  urine,  however, 
contains  very  little  urobilin.  The  actual  body  present  is  a  chromogen 
or  mother  substance  called  urobilinogen,  which  by  oxidation  (such  as 
occurs  when  the  urine  stands  exposed  to  the  air)  is  converted  into 
the  pigment  proper.  In  certain  diseased  conditions  the  amount  of 
urobilin  is  considerably  increased. 

The  most  abundant  urinary  pigment  is  a  yellow  one  called 
urochrome.  It  shows  no  absorption  bands.  It  is  probably  an 
oxidation  product  of  urobilin  (Eiva,  A.  E.  Garrod).  (See  Lesson 
XXVI.) 

Reaction. — The  reaction  of  normal  urine  is  acid.     This  is  not  due 


144 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


to  free  acid,  as  the  uric  and  hippuric  acids  in  the  urine  are  combined 
as  urates  and  hippurates  respectively.  The  acidity  is  due  to  acid  salts, 
especially  acid  sodium  phosphate.  In  certain  circumstances  the 
urine  becomes  less  acid  and  even  alkahne  ;  the  most  important  of 
these  are  as  follov^s  : — 

1.  During  digestion.  Here  there  is  a  formation  of  free  acid  in  the 
stomach,  and  a  corresponding  liberation  of  bases  in  the  blood  which 
passing  into  the  urine  diminish  its  acidity,  or  even  render  it  alkaline. 
This  is  called  the  alkaline  tide ;  the  opposite  condition,  the  acid  tide, 
occurs  after  a  fast — for  instance,  before  breakfast. 

2.  In  herbivorous  animals  and  vegetarians.  The  food  here  con- 
tains excess  of  alkahne  salts  of  acids  such  as  tartaric,  citric,  malic,  &c. 


Fici.  42. — Urinometer  floating  iii  urine 
in  a  testing  glass. 


Fig.  43.— Crystals  of  urea  :  a,  four-sidai 
prisms ;  fc,  indefinite  crystals,  such 
as  are  usually  formed  from  alcohol 
solutions. 


These  acids  are  oxidised  into  carbonates,  which  passing  into  the  urine 
give  it  an  alkaline  reaction. 

Specific  Gravity. — This  should  be  taken  in  a  sample  of  the  twenty- 
four  hours'  urine  with  a  good  urinometer  (see  fig.  42). 

The  specific  gravity  varies  inversely  as  the  quantity  of  urine 
passed  under  normal  conditions  from  1015  to  1025.  A  specific 
gravity  below  1010  should  excite  suspicion  of  hydruria;  one  over 
1030  of  a  febrile  condition,  or  diabetes,  a  disease  in  which  it  may  rise 
to  1050.  The  specific  gravity  has,  however,  been  known  to  sink  as 
low  as  1002  (after  large  potations,  urina  potus),  or  to  rise  as  high  as 
1035  (after  great  sweating)  in  perfectly  healthy  persons. 

Composition. — The  following  table  gives  the  average  amounts 
of  the  urinary  constituents  passed  by  a  man   (taking  an  ordinary 


IFliINK 


145 


diet  containing  about  100  grammes  of  protein)  in  the  twenty-four 
hours : — 


Water 

Total  solids    . 

Urea  . 

Uric  acid 

Hippuric  acid 

Creatinine 

Pigment  and  other  organic  substances 

Sulphuric  acid 

Phosphoric  acid 

Chlorine 

Ammonia 

Potassium 

Sodium 

Calcium 

Magnesium    . 


1500'00  grammes 
72-00 
33-18 

0-55 

0-40 

0-91 
10-00 

201 

3-16 

7-50 

0-77 

2-50 
11-09 

0-26 

0-21 


The  most  abundant  constituents  of  the  urine  are  water,  urea,  and 
sodium  chloride.  In  the  foregoing  table  the  student  must  not  be 
misled  by  seeing  the  names  of  the  acids  and  metals  separated.  The 
acids  and  the  bases  are  combined  to  form  salts  : — urates,  chlorides, 
sulphates,  phosphates,  &c. 

UREA 

Urea  or  Carbamide,  CO(NH.2)2,  is  isomeric  (that  is,  has  the  same 
empirical,  but  not  the  same  structural  formula)  with  ammonium 
cyanate  (NH,)CNO,  from  which  it  was  first  prepared  synthetically 
by  Wohler  in  1828.  Since  then  it  has  been  prepared  synthetically 
in  other  ways.  Wohler's  observation  derives  interest  from  the  fact 
that  this  was  the  first  organic  substance  which  was  prepared  syntheti- 
cally by  chemists. 

When  separated  from  the  urine,  it  is  found  to  be  readily  soluble 
both  in  water  and  in  alcohol  :  it  has  a  saltish  taste,  and  is  neutral  to 
litmus  paper.     The  form  of  its  crystals  is  shown  in  fig.  43. 

When  treated  with  nitric  acid,  nitrate  of  urea  (CON2H4.HNO3) 
is  formed ;  this  crystallises  in  octahedra,  lozenge-shaped  tablets,  or 
hexagons  (fig.  44,  a).  .  When  treated  with  oxalic  acid,  prismatic 
crystals  of  urea  oxalate  (CON2H4.H2C2O4  +  H2O)  are  formed  (fig. 
44,  b). 

These  crystals  may  be  readily  obtained  in  an  impure  form  by 
adding  excess  of  the  respective  acids  to  urine  which  has  been  con- 
centrated to  a  third  or  a  quarter  of  its  bulk.^ 

Under  the  influence  of  certain  organised  ferments,  such  as  the 
micrococcus  ureae,  which  grows  readily  in  stale  urine,  urea  takes  up 

'  The  preparation  of  urea  nitrate  and  urea  oxalate  is  postponed  to  the  next 
lesson,  when  other  microscopic  crystals  will  also  be  under  examination. 


146 


ESSENTIALS   OF  CHEMICAL   PIIYSIOLOCrY 


water,  and  is  converted  into  ammonium  carbonate  [CON2H4  +  2H2O 
=  (NH4)2C03].     Hence  the  ammoniacal  odour  of  putrid  urine. 

By  means  of  nitrous  acid,  urea  is  broken  up  into  carbonic  acid, 
water,  and  nitrogen,  CON2H4  +  2HN02=C02  +  3H20  +  2N2.  This 
may  be  used  as  a  test  for  urea.  Add  fuming  nitric  acid  {i.e.  nitric  acid 
containing  nitrous  acid  in  solution)  to  a  solution  of  urea,  or  to  urine  ; 
an  abundant  evolution  of  gas  bubbles  takes  place. 

Hypobromite  of  soda  decomposes  urea  in  the  following  way : — 

CON2H4  +  3NaBrO  =  CO2  +  N2  +  2H2O  +  3NaBr 


[urea] 


[sodium 
liypobromLte] 


[carbouic 
acid] 


[nitro- 
gen] 


[water]  [sodium 

bromide] 


This  reaction  is  important,  for  on  it  one  of  the  readiest  methods  for 
estimating  urea  depends.  There  have  been  various  pieces  of  appa- 
ratus invented  for  rendering  the  analysis  easy ;  but  the  one  described 


Fig.  44.— a,  nitrate ;  5,  oxalate  of  urea. 

in  the  practical  exercise  at  the  head  of  this  lesson  is  the  best.  If 
the  experiment  is  performed  as  directed,  nitrogen  is  the  only  gas  that 
comes  off,  the  carbonic  acid  being  absorbed  by  excess  of  soda.  The 
amount  of  nitrogen  is  a  measure  of  the  amount  of  urea. 

The  quantity  of  urea  excreted  is  somewhat  variable,  the  chief  cause 
of  variation  being  the  amount  of  protein  food  ingested.  In  a  man 
taking  the  usual  Voit  diet  containing  about  100  grammes  of  protein 
(which  will  contain  about  16  grammes  of  nitrogen)  the  quantity  of 
urea  excreted  daily  averages  33  grammes  (500  grains).  The  per- 
centage in  human  urine  would  then  be  2  per  cent. ;  but  this  also 
varies,  because  the  concentration  of  the  urine  varies  considerably  in 
health.  The  excretion  of  urea  is  usually  at  a  maximum  three  hours 
after  a  meal,  especially  after  a  meal  rich  in  protein. 

Muscular  exercise  has  but  little  effect  on  the  amount  of  nitrogen 


URINE  147 

discharged.  This  is  strikingly  different  from  what  occurs  in  the  case 
of  carbonic  acid  ;  the  more  the  muscles  work,  the  more  carbonic  acid 
do  they  send  into  venous  blood,  which  is  rapidly  discharged  by  the 
expired  air.  Muscular  energy  is  derived  normally  from  the  combus- 
tion of  non-nitrogenous  material ;  this  is  very  largely  carbohydrate. 
If  the  muscles,  however,  are  not  supplied  with  the  proper  amount  of 
carbohydrate  and  fat,  or  if  the  work  done  is  very  excessive,  then  they 
consume  some  of  their  more  precious  protein  material. 

Where  is  Urea  formed  ?— -The  older  authors  considered  that  it 
was  formed  in  the  kidneys,  just  as  they  also  erroneously  thought  that 
carbonic  acid  was  formed  in  the  lungs.  Prevost  and  Dumas  were  the 
first  to  show  that  after  complete  extirpation  of  the  kidney  the  forma- 
tion of  urea  and  other  waste  products  goes  on,  and  these  accumulate 
in  the  blood  and  tissues.  Similarly,  in  those  cases  of  disease  in 
w^hich  the  kidneys  cease  work,  urea  is  still  formed  and  accumulates. 
This  condition  is  called  urcemia  (or  urea  in  the  blood),  and  unless  the 
waste  substances  are  discharged  from  the  body  the  patient  dies. 

UrcBmia. — This  term  was  originally  applied  on  the  erroneous  supposition 
that  it  is  urea  or  some  antecedent  of  urea  which  acts  as  the  poison.  There 
is  no  doubt  that  the  poison  is  not  any  constituent  of  normal  urine  ;  if  the 
kidneys  of  an  animal  are  extirpated  the  animal  dies  in  a  few  days,  but  there 
are  no  uraemic  convulsions.  In  man  also,  if  the  kidneys  are  healthy  or 
approximately  so,  and  suppression  of  urine  occurs  from  the  simultaneous 
blocking  of  both  renal  arteries  by  clot,  or  of  both  ureters  by  stones,  again 
uraimia  does  not  follow.  On  the  other  hand,  uraemia  may  occur  even  while 
a  patient  with  diseased  kidneys  is  passing  a  considerable  amount  of  urine. 
What  the  poison  is  that  is  responsible  for  the  convulsions  and  coma  is 
unknown.  It  is  doubtless  some  abnormal  katabolic  product,  but  whether 
this  is  produced  by  the  diseased  kidney  cells,  or  in  some  other  part  of  the 
body,  is  also  unknown. 

Where,  then,  is  the  seat  of  urea  formation  ?  The  facts  of  experi- 
ment and  of  pathology  point  very  strongly  in  support  of  the  theory 
that  urea  is  formed  in  the  liver.     The  principal  are  the  following  : — 

1.  After  removal  of  the  liver  in  such  animals  as  frogs,  urea  forma- 
tion almost  ceases,  and  ammonia  is  found  in  the  urine  instead. 

2.  In  mammals,  the  extirpation  of  the  liver  is  such  a  serious 
operation  that  the  animals  die.  But  the  liver  of  mammals  can  be  very 
largely  thrown  out  of  gear  by  the  operation  known  as  Eck's  fistula, 
which  consists  in  connecting  the  portal  vein  directly  to  the  inferior 
vena  cava.  In  these  circumstances  the  liver  receives  blood  only 
by  the  hepatic  artery.  The  amount  of  urea  is  lessened,  and  its  place 
is  taken  by  ammonia. 

3.  When  degenerative  changes  occur  in  the  liver,  as  in  cirrhosis  of 
that  organ,  the  urea  formed  is  much  lessened,  and  its  place  is  taken 
by  ammonia.     In  ac2ite  yelloiv  atrophy,  urea  is  almost  absent  from  the 

L    2 


1  48  ESSENTIALS   OF  CHEMICAL  PHYSTO^,OGY 

urine,  and,  again,  there  is  considerable  increase  in  the  ammonia.  In 
this  disease  amino-acids  such  as  leucine  and  tyrosine  are  also  found 
in  the  urine  ;.  these  originate  in  the  intestine,  and,  escaping  further 
decomposition  in  the  degenerated  liver,  pass  as  such  into  the  urine. 

We  have  to  consider  next  the  intermediate  stages  between  protein 
and  urea.  In  order  that  the  student  may  grasp  the  meaning  of  urea 
formation  it  would  be  advisable  for  him  to  turn  again  to  p.  52  and 
read  the  paragraph  there  relating  to  Chittenden's  views  on  diet,  and 
to  pp.  96  to  98,  which  treat  of  protein  absorption,  for  the  question, 
What  is  a  normal  diet  ?  is  intimately  bound  up  with  the  question, 
What  is  a  normal  urine  ?  If,  for  instance,  the  diet  of  the  future  is  to 
contain  only  half  as  much  protein  as  in  the  past,  the  urine  of  the 
future  will  naturally  show  a  nitrogenous  output  of  half  of  that  which 
has  hitherto  been  regarded  as  normal.  In  people  on  such  a  reduced 
diet,  Folin  has  shown  that  the  decrease  in  urinary  nitrogen  falls 
mainly  on  the  urea,  but  certain  other  nitrogenous  katabolites,  parti- 
cularly one  called  creatinine,  remain  remarkably  constant  in  absolute 
amount  in  spite  of  the  great  reduction  in  the  protein  ingested. 

The  laws  governing  the  composition  of  urine  are  obviously  the 
effect  of  the  laws  that  govern  protein  katabolism.  Many  years  ago 
Voit  supposed  that  the  protein  ingested  was  utilised  partly  in  tissue 
formation,  and  partly  remained  in  the  circulating  fluids  as  '  circulating 
protein  ' ;  he  further  considered  that  the  breakdown  of  the  protein  in 
the  tissues  was  accomplished  with  much  greater  difficulty  than  that 
in  blood  and  lymph,  and  that  the  small  amount  of  '  tissue-protein ' 
which  disintegrates  as  the  result  of  the  wear  and  tear  of  the  tissues 
was  dissolved  and  added  to  the  '  circulating  protein,'  in  which  alone 
the  formation  of  final  katabolic  products  such  as  urea  was  supposed 
to  occur.  As  time  went  on,  it  was  shown  that  many  facts  were 
incompatible  with  this  theory,  and  so  it  was  largely  displaced  by 
Pfliiger's  view,  in  which  it  was  held  that  the  food  protein  must  first 
be  assimilated,  and  become  part  and  parcel  of  living  cells,  before 
katabolism  occurs.  We  now  know  that  neither  of  these  views  is 
correct,  and  that  nitrogenous  katabolism  is  of  two  kinds  :  one  kind 
varies  with  the  food ;  it  is  therefore  variable  in  amount,  and  occurs 
almost  immediately  or  within  a  few  hours  after  the  food  is  absorbed  ; 
the  amino-acids  absorbed  from  the  intestine  are  in  great  measure  never 
built  into  living  protoplasm  at  all,  and  are  simply  taken  to  the  liver, 
where  they  are  converted  into  urea.  This  variety  of  katabolism  is 
called  exogenous.  The  other  kind  of  metabolism  is  constant  in  quantity 
and  smaller  in  amount,  and  is  due  to  the  actual  breakdown  of  protein 
matter  in  the  body  cells  and  tissues,  which  had  been  built  into  them 


URINE  149 

previously.  This  form  of  metabolism  is  called  endogenous  or  tissue 
metabolism,  and  the  final  product  is  not  urea  to  any  great  extent,  but 
the  waste  nitrogen  finds  its  way  out  of  the  body  in  other  substances, 
of  which  creatinine  appears  to  be  the  most  important.  This  form  of 
metabolism  sets  a  limit  to  the  lowest  level  of  nitrogenous  requirement 
attainable  ;  the  protein  sufficient  to  maintain  it  is  indispensable- 
Whether  the  amount  of  protein  which  is  exogenously  metabolised 
can  be  entirely  dispensed  with  is  at  present  questionable,  and  those 
who  seek  to  replace  it  entirely  by  non-nitrogenous  food  are  living 
dangerously  near  the  margin.  A  point  of  considerable  importance  in 
this  connection  is,  that  the  nitrogen  of  the  protein  is  split  off  from  it 
by  hydrolysis  without  oxidation.  There  is  thus  very  little  loss  of 
potential  energy,  the  energy  of  the  products  being  nearly  equal  to 
that  of  the  original  protein ;  it  is,  however,  the  non-nitrogenous 
residue  which  is  mainly  available  for  oxidation,  and  thus  for  calorific 
processes.  The  fact  that  muscular  work  does  not  normally  increase 
nitrogenous  metabolism  becomes  intelligible  in  the  light  of  the  con- 
sideration that  protein  katabolism,  in  so  far  as  its  nitrogen  is  concerned, 
is  independent  of  the  oxidations  which  give  rise  to  heat,  or  to  the 
energy  which  is  converted  into  work.  Those  who  in  the  past  have 
endeavoured  to  study  the  relation  of  muscular  work  to  nitrogen 
excretion  have  usually  estimated  the  urea.  Now  that  we  know  urea 
is  the  chief  end  product  of  exogenous  and  not  of  tissue  katabolism, 
we  see  that  estimations  of  urea  can  give  us  but  little  real  information 
on  this  point.  The  substance  which  ought  to  be  estimated  is 
creatinine,  and  it  has  been  found  recently  that  even  this  is  not  notably 
increased  in  the  urine,  provided  the  muscles  receive  their  normal 
supply  of  fat  and  carbohydrate.  The  body  is  very  economical  in  so 
far  as  protein  is  concerned,  and  tissue  or  endogenous  katabolism  is 
kept  at  a  low^  level. 

What  is  the  proportion  between  exogenous  and  endogenous 
nitrogen  katabolism  ?  It  is  very  difficult  to  give  any  exact  estimate. 
We  do  know  that  in  ordinary  diets,  the  former  is  far  in  excess,  and 
probably  in  a  man  excreting  16  grammes  of  nitrogen  daily  (that  is, 
the  amount  corresponding  to  an  intake  of  100  grammes  of  protein), 
only  a  quarter  of  this  or  even  less  represents  tissue  breakdown. 

The  view  we  have  advanced  concerning  urea  formation,  then,  is, 
that  it  is  mainly  the  result  of  the  conversion,  by  the  liver,  of  amino- 
acids  absorbed  from  the  intestine  into  that  substance.  This  view 
receives  confirmation  from  experiments  in  which  certain  amino-acids, 
such  as  glycine,  leucine,  and  arginine,  have  been  injected  direct  into 
the  blood-stream.     The  result  is  an  increased  formation  of  urea.     In 


150  ESSENTIALS   OF   CHEMICAL  PHYSIOLOGY 

the  case  of  arginine  the  exact  chemical  decomposition  which  takes 
place  is  known.  We  have  already  seen  that  arginine  is  a  compound 
of  a  urea  radical  and  a  substance  called  ornithine  (di-amino-valeric 
acid,  see  p.  32)  ;  the  liver  is  able  to  hydrolyse  arginine,  and  so  urea 
is  liberated.  This  power  is  due  to  the  action  of  a  special  ferment 
called  arginase,  which,  although  it  is  also  found  in  other  organs,  is 
specially  abundant  in  the  liver.  It  is,  however,  possible  that  the 
ornithine  itself  may  be  further  broken  up,  and  so  an  extra  quantity  of 
urea  formed.  On  the  other  hand  there  are  some  amino-acids  {e.g. 
tyrosine)  which  on  injection  do  not  lead  to  any  increase  in  urea 
formation. 

If,  however,  we  glance  at  the  formula  of  ornithine,  we  see  that  it 
has  one  point  in  common  with  other  amino-acids,  such  as  glycine  and 
leucine,  to  take  simple  examples  : — 

Ornithine     05lI,2N2O2 
Glycine        C2H5NO2 
Leucine       C6H13NO2 

This  is,  that  in  all  cases  the  carbon  atoms  are  more  numerous  than 
the  nitrogen  atoms.  In  urea,  CON2H4,  the  reverse  is  the  case.  The 
amino-acids  must  therefore  be  split  into  simpler  compounds,  which 
unite  with  one  another  to  form  urea.  Urea  formation  is  thus  in 
part  synthetic.  These  simpler  compounds  are  ammonium  salts. 
Schroder's  work  proves  that  ammonium  carbonate  is  one  of  the 
urea  precursors,  if  not  the  principal  one.  The  equation  which 
represents  the  reaction  is  as  follows  : — 

(NH4)2C03=CON2H4  +  2H2O 

[ammouiam  [urea]  [water] 

carbonate] 

Schroder's  principal  experiment  was  this :  a  mixture  of  defibrinated 
blood  and  ammonium  carbonate  was  injected  into  the  liver  by  the 
portal  vein  ;  the  blood  leaving  the  liver  by  the  hepatic  vein  was  found 
to  contain  urea  in  great  abundance.  This  does  not  occur  when  the 
same  experiment  is  performed  with  any  other  organ  of  the  body,  so 
that  Schroder's  experiments  also  prove  the  great  importance  of  the 
liver  in  urea  formation.  Similar  results  were  obtained  by  Nencki 
with  ammonium  carbamate. 

We  must  further  remember  that  ammonia  itself  is  one  of  the 
products  of  digestion  of  protein  in  the  intestine,  and  it  may  possibly, 
to  a  small  extent,  be  a  result  of  tissue  katabolism.  This  ammonia 
passes  into  the  blood,  where  it  unites  with  carbonic  acid  to  form  either 
the  carbamate  or  carbonate  of  ammonium.     Thus  ammonia,  whether 


UKINE  151 

formed  directly  or  by  the  breakdown  of  amino-acids,  is  the  principal 
immediate  precursor  of  urea. 

The  following  structural  formulae  show  the  relationship  between 
ammonium  carbonate,  ammonium  carbamate,  and  urea : — 

o-r/ONH4         o-r/NH2  o-r/NH2 

"-'^XO.NH^  ^-^XO.NH^  "-^\NH, 

[auimoniuin  carbonate]  [ammouium  carbamate]  [urea  or  carbamide] 

The  loss  of  one  molecule  of  water  from  ammonium  carbonate  pro- 
duces ammonium  carbamate ;  the  loss  of  a  second  molecule  of  water 
produces  urea. 

AMMONIA 

The  urine  of  man  and  carnivora  contains  small  quantities  of 
ammonium  salts.  The  reason  that  some  ammonia  always  slips 
through  into  the  urine  is  that  a  part  of  the  ammonia-containing 
blood  passes  through  the  kidney  before  reaching  organs,  such  as  the 
liver,  which  are  capable  of  synthesising  urea.  In  man  the  daily 
amount  of  ammonia  excreted  varies  between  0*3  and  1*2  gramme : 
the  average  is  0*7  gramme.  The  ingestion  of  ammonium  carbonate 
does  not  increase  the  amount  of  ammonia  in  the  urine,  but  increases 
the  amount  of  urea,  into  which  substance  the  ammonium  carbonate 
is  easily  converted.  But  if  a  more  stable  salt,  like  ammonium 
chloride,  is  given,  it  appears  as  such  in  the  urine. 

Under  normal  circumstances  the  amount  of  ammonia  depends  on 
the  adjustment  between  the  production  of  acid  substances  in  meta- 
bolism and  the  supply  of  bases  in  the  food.  Ammonia  formation  is 
the  physiological  remedy  for  deficiency  of  bases. 

When  the  production  of  acids  is  excessive  (as  in  diabetes),  or 
when  mineral  acids  are  given  by  the  mouth  or  injected  into  the 
blood-stream,  the  result  is  an  increase  of  the  physiological  remedy, 
and  excess  of  the  ammonia  passes  over  into  the  urine.  Under  normal 
conditions  ammonia  is  kept  at  a  minimum,  being  finally  converted 
into  the  less  toxic  substance  urea,  which  the  kidneys  easily  excrete. 
The  defence  of  the  organism  against  acids  which  are  very  toxic  is  an 
increase  of  ammonia  formation,  or,  to  put  it  more  correctly,  less  of 
the  ammonia  formed  is  converted  into  urea. 

Under  the  opposite  conditions — namely,  excess  of  alkali,  either  in 
food  or  given  as  such — the  ammonia  disappears  from  the  urine,  all 
being  converted  into  urea.  Hence  the  diminution  of  ammonia  in  the 
urine  of  man  on  a  vegetable  diet,  and  its  absence  in  the  urine  of 
herbivorous  animals. 

Not  only  is  this  the  case  but  if  ammonium  chloride  is  given  to  a 


152  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

herbivorous  animal  like  a  rabbit,  the  urinary  ammonia  is  but  little 
increased.  It  reacts  with  sodium  carbonate  in  the  tissues,  forming 
ammonium  carbonate  (which  is  excreted  as  urea)  and  sodium  chloride. 
Herbivora  also  suffer  much  more  from,  and  are  more  easily  killed  by, 
acids  than  carnivora,  their  organisation  not  permitting  a  ready  supply 
of  ammonia  to  neutralise  excess  of  acids. 

CREATININE 

Creatinine  is  one  of  the  substances  in  the  urine  which  represents 
the  end-stage  of  the  tissue  or  endogenous  katabolism  of  protein.  It 
is  the  most  abundant  of  such  katabolites,  and  the  one  concerning 
which  we  know  most. 

The  most  abundant  of  our  active  tissues  is  muscle,  and  the 
amount  of  nitrogenous  waste  in  this  tissue  which  leaves  it  as  urea 
is  insignificant.  The  place  of  urea  is  taken  by  another  substance 
called  creatine.  As  already  explained  on  p.  32,  creatine  is  a  com-' 
pound  of  a  urea  radical  with  another  group,  and  when  it  is  boiled 
with  baryta  water  it  takes  up  water  and  splits  into  two  substances 
— namely,  urea  and  sarcosine  or  methyl-glycine  ;  this  is  shown  in 
the  following  equation  : — 

^'"^     N(CH3)CH2.COOH  +  H20=H2N/^^ 


[creatiue]  [water]  [urea] 

+  NH.CH3.CH2.COOH 

[sarcosine] 

It  is,  however,  extremely  doubtful  whether  this  decomposition  occurs 
in  the  body,  and  therefore  whether  creatine  is  to  any  important  degree 
a  precursor  of  urea.  For,  when  creatine  is  introduced  directly  into 
the  blood-stream,  the  amount  of  urea  is  not  increased  in  the  urine. 
What  is  increased  is  another  substance  called  creatinine,  which  is 
creatine  minus  wdter,  as  shown  in  the  following  equation  : — 
C4H9N3O2        =C4H7N30         +U,0 

[creatine]  [creatinine]  [water] 

Creatinine,  therefore,  comes  from  nitrogenous  katabolism  in  the 
tissues,  especially  muscular  tissues,  and  creatine  is  an  intermediate 
stage  in  its  formation.  Some  of  the  creatinine,  however,  has  a 
different  origin — namely,  an  exogenous  one ;  that  is,  from  the  food 
instead  of  from  tissue  metabolism ;  and  the  substance  in  the  food 
which  gives  rise  to  it  is  the  creatine  contained  in  the  flesh  eaten. 

The  best  method  for  estimating  creatinine  and  creatine  is  given  in 
Lesson  XXV. 


URINE  168 


THE  INORGANIC   CONSTITUENTS   OF  URINE 

The  inorganic  or  mineral  constituents  of  urine  are  chiefly  chlorides, 
phosphates,  sulphates,  and  carbonates ;  the  metals  with  which  these 
are  in  combination  are  sodium,  potassium,  ammonium,  calcium,  and 
magnesium.  The  total  amount  of  these  salts  excreted  varies  from  19 
to  25  grammes  daily.  The  most  abundant  is  sodium  chloride,  which 
averages  in  amount  10  to  13  grammes  per  diem.  These  substances 
are  derived  from  two  sources — first  from  the  food,  and  secondly  as  the 
result  of  metabolic  processes.  The  chlorides  and  most  of  the  phos- 
phates come  from  the  food ;  the  sulphates  and  some  of  the  phosphates 
are  a  result  of  metabolism.  The  sulphates  are  derived  from  the 
changes  that  occur  in  the  proteins  ;  the  nitrogen  of  proteins  leaves  the 
body  chiefly  as  urea ;  the  sulphur  of  the  proteins  is  oxidised  to  form 
sulphuric  acid,  which  passes  into  the  urine  in  the  form  of  sulphates. 
The  excretion  of  sulphates,  moreover,  runs  parallel  to  that  of  urea. 
Sulphates,  like  urea,  are  the  result  of  exogenous  protein  metabolism ; 
endogenous  metabolism  so  far  as  sulphur  is  concerned  is  represented 
in  the  urine  partly  as  ethereal  sulphates,  but  chiefly  by  less  fully 
oxidised  compounds  of  sulphur.  The  chief  tests  for  the  various  salts 
have  been  given  in  the  practical  exercises  at  the  head  of  this  lesson. 

Chlorides. — The  chief  chloride  is  that  of  sodium.  The  ingestion  of 
sodium  chloride  is  followed  by  its  appearance  in  the  urine,  some  on  the 
same  day,  some  on  the  next  day.  Some  is  decomposed  to  form  the 
hydrochloric  acid  of  the  gastric  juice.  The  salt,  in  passing  through 
the  body,  fulfils  the  useful  office  of  stimulating  metabolism  and 
excretion. 

Sulphates. — The  sulphates  in  the  ludne  are  principally  those  of 
potassium  and  sodium.  They  are  derived  from  the  metabolism  of 
proteins  in  the  body.  Only  the  smallest  trace  enters  the  body  with  the 
food.  Sulphates  have  an  unpleasant  bitter  taste  (for  instance,  Epsom 
salts) ;  hence  we  do  not  take  food  that  contains  them.  The  sulphates 
vary  in  amount  from  1*^5  to  3  grammes  daily. 

In  addition  to  these  sulphates  there  is  a  small  quantity  of 
sulphuric  acid  comprising  about  one-tenth  of  the  total  which 
is  combined  with  organic  radicals ;  the  compounds  are  known  as 
ethereal  sulphates,  and  they  originate  mainly  from  putrefactive 
processes  occurring  in  the  intestine.  The  most  important  of  these 
ethereal  sulphates  are  phenyl  sulphate  of  potassium  and  indoxyl 
sulphate  of  potassium.  The  latter  originates  from  the  indole  formed 
ill  the  intestine,  and   as  it   yields  indigo   when  treated  with  certain 


154  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

reagents  it  is  sometimes  called  indican.  It  is  very  important  to 
remember  that  the  indican  of  urine  is  not  the  same  thing  as  the 
indican  of  plants.  Both  yield  indigo,  but  there  the  resemblance 
ceases. 

The  equation  representing  the  formation  of  potassium  phenyl- 
sulphate  is  as  follows  : — 

CH^OH  +  SO^/Of  =  SO,/0^«H,^jj^O 

[phenol]  [potassium  [potassium  [water] 

hydrogen  sulphate]  phenyl-sulphate] 

The  formation  of  potassium  indoxyl-sulphate  may  be  represented 

CH 

as  follows  : —  Indole,  C6H4/  ^CH  on  absorption  is  converted 

NH 
O.OH 

intoindoxyl:     CgH/^CH 

NH 
Indoxyl   then   interacts   with   potassium    hydrogen   sulphate   as 
follows  : — 

[indoxyl]  [potassium  [potassium  [water] 

hydrogen  sulphate]        iudoxyl-sulphate] 

The  formation  of  such  sulphates  is  important ;  the  aromatic  sub- 
stances liberated  by  putrefactive  processes  in  the  intestine  are 
poisonous,  but  their  conversion  into  ethereal  sulphates  renders  them 
innocuous.  (For  tests  for  indoxyl  in  urine  see  Advanced  Course, 
Lesson  XXVI.) 

Carbonates. — Carbonates  and  bicarbonates  of  sodium,  calcium, 
magnesium,  and  ammonium  are  present  in  alkaline  urine  only.  They 
arise  from  the  carbonates  of  the  food,  or  from  vegetable  acids  (malic, 
tartaric,  &c.)  in  the  food.  They  are,  therefore,  found  in  the  urine  of 
herbivora  and  vegetarians,  whose  urine  is  thus  rendered  alkaline. 
Urine  containing  carbonates  becomes,  like  saliva,  cloudy  on  stand- 
ing, the  precipitate  consisting  of  calcium  carbonate,  and  also  phos- 
phates. 

Phosphates. — Two  classes  of  phosphates  occur  in  normal 
urine  : — 

(1)  Alkaline  phosphates — that  is,  phosphates  of  sodium  (abundant) 
and  potassium  (scanty). 


URINE 


165 


(2)  Earthy  phosphates — that  is,  phosphates  of  calcium  (abundant) 
and  magnesium  (scanty). 

The  composition  of  the  phosphates  in  urine  is  hable  to  variation. 
In  acid  urine  the  acidity  is  due  to  the  acid  salts.  These  are  chiefly 
sodium  dihydrogen  phosphate,  NaH2P04,  and  calcium  dihydrogen 
phosphate,  Ca(H2P04)2. 

In  neutral  urine,  in  addition,  disodium  hydrogen  phosphate 
(Na2HP04),  calcium  hydrogen  phosphate,  CaHP04,  and  magnesium 
hydrogen  phosphate,  MgHP04,  are  found.  In  alkahne  urine 
there  may  be  instead  of,  or  in  addition  to  the  above,  the  normal 
phosphates  of  sodium,  calcium,  and  magnesium  [Na3P04,  Ca3(P04)2, 

Mg3(P04)2]. 

The  earthy  phosphates  are  precipitated  by  rendering  the  urine 
alkaline  by  ammonia.  In  decomposing  urine,  ammonia  is  formed 
from  the  urea  :  this  also  precipitates  the  earthy  phosphates.  The 
phosphates  most  frequently  found  in  the  white  creamy  precipitate 
which  occurs  in  decomposing  urine  are — 

(1)  Triple  phosphate  or  ammonio-magnesium 
phosphate  (NH4MgP04  +  6H20).  This  crystallises 
in  '  coffin-lid '  crystals  (see  fig.  45)  or  feathery  stars. 

(2)  Stellar  phosphate,  or  calcium  phosphate, 
which  crystallises  in  star-like  clusters  of  prisms. 

As  a  rule,  normal  urine  gives  no  precipitate  when 
it  is  boiled ;  but  sometimes  neutral,  alkaline,  and 
occasionally  faintly  acid  urines  give  a  precipitate  of 
calcium  phosphate  when  boiled  ;  this  precipitate  is 
amorphous,  and  is  liable  to  be  mistaken  for  albumin. 
It  may  be  distinguished  readily  from  albumin,  as  it 
is  soluble  in  a  few  drops  of  acetic  acid,  whereas 
coagulated  protein  does  not  dissolve. 

The  phosphoric  acid  in  the  urine  chiefly  originates  from  the  phos- 
phates of  the  food,  but  is  partly  a  decomposition  product  of  thephos- 
phorised  organic  materials  in  the  body,  such  as  lecithin  and  nuclein. 
The  amount  of  P2O5  in  the  twenty-four  hours'  urine  varies  from  2"5 
to  3*5  grammes,  of  which  the  earthy  phosphates  contain  about  half 
(1  to  1-5  gr.). 


Fig.  45.  —  Ammouio- 
magnesium  or  triple 
phosphate. 


LESSON   XI 
UBINE  {contmued) 

1.  Urea  Nitrate.  —Evaporate  some  urine  in  a  capsule  to  a  quarter  of  its 
bulk.  Pour  the  concentrated  urine  into  a  watch-glass ;  let  it  cool,  and  add  a 
few  drops  of  strong,  but  not  fuming,  nitric  acid.  Crystals  of  urea  nitrate 
separate  out.     Examine  these  microscopically. 

2.  Urea  Oxalate. — Concentrate  the  m-ine  as  in  the  last  exercise,  and  add 
oxalic  acid.  Crystals  of  urea  oxalate  separate  out.  Examine  these  micro- 
scopically. 

3.  Uric  Acid. — Examine  microscopically  the  crystals  of  uric  acid  in  some 
urine,  to  which  5  per  cent,  of  hydrochloric  acid  has  been  added  twenty-four 
hours  previously.  Note  that  they  are  deeply  tinged  with  pigment,  and  to  the 
naked  eye  look  like  granules  of  cayenne  pepper. 

When  microscopically  examined,  the  crystals  are  seen  to  be  large  bundles, 
principally  in  the  shape  of  barrels,  with  spicules  projecting  from  the  ends, 
and  whetstones.  If  oxalic  acid  is  used  instead  of  hydrochloric  acid  in  this 
experiment,  the  crystals  are  smaller,  and  more  closely  resemble  those  observed 
in  pathological  urine  in  cases  of  uric  acid  gravel  (see  fig.  46). 

Dissolve  the  crystals  in  caustic  potash  and  then  carefully-  add  excess  of 
hydrochloric  acid.     Small  crystals  of  uric  acid  again  form. 

Murexide  Test. — Place  a  little  uric  acid,  or  a  urate  (for  instance,  serpent's 
urine),  in  a  capsule  ;  add  a  little  dilute  nitric  acid  and  evaporate  to  dryness. 
A  yellowish-red  residue  is  left.  Add  a  little  ammonia  carefully.  The  residue 
turns  to  violet.  This  is  due  to  the  formation  of  nuirexide  or  purpurate  of 
ammonia.     On  the  addition  of  potash  the  colour  becomes  bluer. 

Schiff's  Test. — Dissolve  some  uric  acid  in  sodium  carbonate  solution. 
Put  a  drop  of  this  on  blotting  pax)er,  add  a  drop  of  silver  nitrate,  and  warm 
gently ;  the  black  colour  of  reduced  silver  is  seen  on  the  paper. 

4.  Deposits  of  Urates  or  Lithates  (Lateritious  Deposit). — The  specimen  of 
urine  from  the  hospital  contains  excess  of  urates,  which  have  become  deposited 
on  the  urine  becoming  cool.  They  are  tinged  with  pigment  (uroerythrin), 
and  have  a  pinkish  colour,  like  brickdust ;  hence  the  term  '  lateritious.' 
Examine  microscopically.  The  deposit  is  usually  amorphous — that  is,  non- 
crystalline. Sometimes  crystals  of  calcium  oxalate  (envelope  crystals — 
octahedra)  are  seen  also ;  these  are  colourless. 

The  deposit  of  urates  dissolves  on  heating  the  urine. 

5.  Deposit  of  Phosphates. — Another  specimen  of  pathological  urine  contains 
excess  of  phosphates,  which  have  formed  a  white  deposit  on  the  urine  be- 
coming alkaline.  This  precipitate  does  not  dissolve  on  heating ;  it  may  be 
increased.  It  is,  however,  soluble  in  acetic  acid.  Examine  microscopically 
for  cofiin-lid  crystals  of  triple  phosphate  (ammonio-magnesium  phosphate), 
for  crystals  of  stellar  (calcium)  phosphate,  and  for  mucus.  Mucus  is  flocculent 
to  the  naked  eye,  amorphous  to  the  microscope. 

N.B. — On  boiling  neutral,  alkaline,  or  even  faintly  acid  urine  it  may  be- 
come turbid  from  deposition  of  phosphates.     The  solubility  of  this  deposit  in 


URINE  157 

a  few  drops  of  acetic  acid  distinguishes  it  from  albumin,  for  which  it  is  liable 
to  be  mistaken. 

Some  of  the  facts  described  in  the  foregoing  exercises  have  been  already- 
dwelt  upon  in  the  preceding  lesson.  They  are,  however,  conveniently  grouped 
together  here,  as  all  involve  the  use  of  the  microscope. 


URIC  ACID 

Uric  Acid  (C5N4H403)is  in  mammals  the  medium  by  which  only  a 
small  quantity  of  nitrogen  is  excreted  from  the  body.  It  is,  however, 
in  birds  and  reptiles  the  principal  nitrogenous  constituent  of  their 
urine.  It  is  not  present  in  the  free  state,  but  is  combined  with  bases 
to  form  urates. 

It  may  be  obtained  from  human  urine  by  adding  5  c.c.  of  hydro- 
chloric acid  to  100  c.c.  of  the  urine,  and  allowing  the  mixture  to  stand 
for  twelve  to  twenty-four  hours.  The  crystals  which  form  are  deeply 
tinged  with  urinary  pigment,  and  though  by  repeated  solution  in 
caustic  soda  or  potash,  and  reprecipitation  ,,^^^    y^^^^ 

l)y  hydrochloric  acid,  they  may  be  obtained  ^^^^^^^^^^^  / — \^^ 

fairly  free  from  pigment,  pure  uric  acid  is  /^^  ^ 

more  readily  obtained  from  the  solid  urine  of     /^  ^^^^^    rj 
a  serpent  or  bird,  which  consists  principally  ^     ^  5^^ 

of  the  acid  ammonium  urate.     This  is  dis-       0  ^-^^ 

solved  in  soda,  and  then  the   addition   of  ^^  fl     %      ^ 

hydrochloric  acid  produces  as  before  the         %    ^      ^p  a 

crystallisation  of  uric  acid  from  the  solution,      f    ^     ^  I — . 

The  pure  acid  crystallises  in  colourless  0         w 

rectangular  plates  or  prisms.  In  striking 
contrast  to  urea  it  is  a  most  insoluble  sub- 
stance, requiring  for  its  solution  1,900  parts 
of  hot  and  15,000  parts  of  cold  water.  The  '''''•  *'-''''"  ''^"'  "'"'"^'• 
forms  which  uric  acid  assumes  when  precipitated  from  human  urine, 
either  by  the  addition  of  hydrochloric  acid  or  in  certain  pathological 
processes,  are  very  various,  the  most  frequent  being  the  whetstone 
shape  ;  there  are  also  bundles  of  crystals  resembling  sheaves,  barrels, 
and  dumb-bells  (see  fig.  46). 

The  murexide  test  which  has  just  been  described  among  the 
practical  exercises  is  the  principal  test  for  uric  acid.  The  test  has 
received  the  name  on  account  of  the  resemblance  of  the  colour  to  the 
purple  of  the  ancients,  which  was  obtained  from  certain  snails  of  the 
genus  Murex. 

Another  reaction  that  uric  acid  undergoes  (though  it  is  not  appli- 
cable as  a  test)  is  that  on  treatment  w4th  certain  oxidising  reagents 


158  ESSENTIALS   OF   CHEMICAL  PHYSIOLOGY 

urea  aud  oxalic  acid  can  be  obtained  from  it.  It  is,  however,  doubtful 
whether  a  similar  oxidation  occurs  in  the  normal  metabolic  processes 
of  the  body. 

Uric  acid  is  dibasic,  and  thus  there  are  two  classes  of  urates — the 
normal  urates  and  the  acid  urates.  A  normal  urate  is  one  in  which 
two  atoms  of  the  hydrogen  are  replaced  by  two  of  a  monad  metal  like 
sodium;  an  acid  urate  is  one  in  which  only  one  atom  of  hydrogen.is 
thus  replaced.     The  formulae  would  be — 

C5H4N40;j  =uric  acid 
C.5H3NaN403=acid  sodium  urate 
05H2Na9N4O3=normal  sodium  urate 

The  acid  sodium  urate  is  the  chief  constituent  of  the  pinkish  deposit 
of  grates,  which,  as  w^e  have  already  stated,  is  called  the  lateritious 
deposit. 

If  uric  acid  is  represesented  by  H^U,  the  normal  urates  may  be  represented 
by  MoU  and  the  acid  urates  by  MHU.  Bence  Jones,  and  later  Sir  W.  Roberts, 
considered  that  the  urates  which  actually  occur  in  urine  are  quadriurates, 
MHU.H^U.  There  is  much  doubt  whether  such  compounds  actually  exist ; 
if  they  do  they  are  readily  decomposed  into  acid  urates  MHU  and  free  uric 
acid,  HoU. 

The  quantity  of  uric  acid  excreted  by  an  adult  varies  from  7  to  10 
grains  (0*5  to  0-75  gramme)  daily. 

The  best  method  for  determining  the  quantity  of  uric  acid  in  the 
urine  is  that  of  Hopkins.  Ammonium  chloride  in  crystals  is  added 
to  the  urine  until  no  more  will  dissolve.  This  saturation  completely 
precipitates  all  the  uric  acid  in  the  form  of  ammonium  urate.  After 
standing  for  two  hours  the  precipitate  is  collected  on  a  filter,  washed 
with  saturated  solution  of  ammonium  chloride,  and  then  dissolved  in 
weak  alkali.  From  this  solution  the  uric  acid  is  precipitated  by 
neutralising  with  hydrochloric  acid.  The  precipitate  of  uric  acid  is 
collected  on  a  weighed  filter,  dried  and  weighed,  or  titration  may  be 
performed  with  potassium  permanganate  (see  Advanced  Course). 

Origin  of  Uric  Acid. — Uric  acid  is  not  made  by  the  kidneys. 
When  the  kidneys  are  removed  uric  acid  continues  to  be  formed  and 
accumulates  in  the  organs,  especially  in  the  liver  and  spleen.  The 
liver  has  been  removed  from  birds,  and  uric  acid  is  then  hardly  formed 
at  all,  its  place  being  taken  by  ammonia  and  lactic  acid.  It  is  there- 
fore probable  that  in  these  animals  ammonia  and  lactic  acid  are 
normally  synthesised  in  the  liver  to  form  uric  acid. 

This  synthetic  origin  of  uric  acid,  which  is  so  important  in  birds 
and  snakes,  does  not,  however,  occur  in  mammals.  In  mammals 
uric  acid  is  the  chief  end-product  of  the  katabolism  of  cell  nuclei  or 


UHINK  159 

of  imclein,  the  principal  constituent  of  the  nuclei.  This  therefore 
leads  us  next  to  study : — 

Purine  Substances. — Emil  Fischer  has  shown  that  the  decom- 
position products  of  nuclein  are  derivatives  of  a  substance  he  has 
named  purine.  The  empirical  formulae  for  purine,  the  purine  bases, 
and  uric  acid  are  as  follows  : — 

Purine       .         .  C5H4N4 

Hypoxanthine  .  C5H4N4O  Monoxypurine      \ 

Xanthine  .         .  C5H4N4O2  Dioxypurine  |  Purine 

Adenine    .         .  C5H3N4.NH2      Amino-purine       I  bases. 

Guanine    .         .  C5H3N4O.NH2  Amino-oxypurine/ 

Uric  acid .         .  C-,H4N403  Trioxypurine 

There  are  a  vast  number  of  purine  derivatives,  but  only  a  few 
of  them  have  at  present  any  physiological  importance.  Others  in 
addition  to  those  already  enumerated  are  theophylline  (dimethyl- 
xanthine),  theobromine  (also  a  dimethy] -xanthine),  caffeine  (trimethyl- 
xanthine) ;  these  are  of  interest,  as  they  occur  in  tea,  cocoa,  and  coffee. 
A  few  words  more  may  be  added  in  respect  to  those  in  our  list. 

Purine  itself  has  never  been  discovered  in  the  body.  It  has  the 
following  structural  formula : 

N=C-H 

I  I 

H  -  C     C  -  NH. 

II  !l  ^C-H 

N  -C-N    ^ 

The  purine  nucleus  is  depicted  in  the  next  formula,  and  its  atoms 
have  been  empirically  numbered  as  shown  for  convenience  of 
description  : — 

IN  -«C 
I  i 


3N  _  4C  -  9N 


/ 


Hypoxanthine  or  Sarcine  is  found  in  the  body  tissues  and  fluids, 
and  in  the  urine.  It  is  derived  from  some  nucleins,  especially 
those  from  fishes'  spermatozoa.  It  may  be  termed  6-oxypurine, 
as  the  oxygen  is  attached  to  the  atom  number  6  in  the  purine 
nucleus. 

Xanthine  is  found  with  hypoxanthine  in  the  body,  and  has  been 
obtained  from  a  number  of   nucleins  (from  spermatozoa,  thymus. 


1C)0  ESSENTIALS  OF  CHEMICAL  PHYSIOLOaY 

pancreas,    &c.).      It   is   2,   6-dioxypurine,   its   oxygen   atoms    being 
attached  to  the  atoms  numbered  2  and  6  in  the  purine  nucleus. 

NH--C=0  NH-C  =  0 

II  II 

H-C         C-NH.  0  =  0        C-NH. 

II  II  >C~H  I  II  >C-H 

[hypoxanthiue]  [xftnthine] 

Adenine  is  found  in  the  tissues,  blood,  and  urine.  It  is  obtained 
from  several  nucleins,  but  especially  from  the  nuclein  derived  from 
the  thymus.     It  is  6-amino-purine. 

Guanine  is  also  a  decomposition  product  of  nucleins,  especially 
of  that  obtained  from  the  pancreas.  Combined  with  calcium  it 
gives  the  brilliancy  to  the  scales  of  fishes,  and  is  also  found  in  the 
bright  tapetum  of  the  eyes  in  these  animals.  It  is  a  constituent  of 
guano,  and  here  is  probably  derived  from  the  fish  eaten  by  marine 
birds.     It  is  2-amino-6-oxypurine. 


H 


N  =  C  -  NH., 

1         1 

NH- 

1 

-C  =  0 

H-C      C  -  NH.                    H2N  - 
II        II              >C-H 

N  -  C  -     W/ 

1 
-C 

II 

N    - 

C-  NH. 

II          >c 

-  C    -    N'^ 

[adenine] 

[guanine] 

Uric  acid  is  2,  6,  8-trioxypurine. 

NH  -  C  =  0 

1            1 

0  =  C          C  -  NH. 

1        II           >co 

NH-C  -  NH/ 

[nric  acid] 

The  close  chemical  relationship  of  uric  acid  to  the  purine  bases  is 
obvious  from  a  study  of  the  formulae  just  given.  Just  as  in  the  case 
of  urea,  uric  acid,  however,  may  be  exogenously  or  endogenously 
formed.  Certain  kinds  of  food  increase  uric  acid  because  they  con- 
tain nuclein  (for  instance,  sweetbreads)  in  abundance,  or  purine  bases 
(for  instance,  hypoxanthiue  in  meat) ;  the  uric  acid  which  originates 
in  this  way  is  termed  exogenous.  Certain  diets,  on  the  other  hand, 
increase  uric  acid  formation  by  leading  to  an  increase  of  leucocytes, 
and  consequently  increase  in  the  metabolism  of  their  nuclei ;  in  other 
cases  the  leucocytes  may  increase  from  other  causes,  as  in  the  disease 
named  leucocythaemia.  The  uric  acid  that  arises  from  nuclear  kata- 
bolism  is  termed  e^idogenous.     Although  special  attention  has  been 


URINE  161 

directed  to  the  nuclei  of  leucocytes  because  they  can  be  readily 
examined  during  life,  it  must  be  remembered  that  nuclein  meta- 
bolism of  all  cells  may  contribute  to  uric  acid  formation. 

A  study  of  uric  acid  formation  forms  a  useful  occasion  on  which 
to  allude  to  ferment  actions  in  metabolism  generally.  Ferments  of 
a  digestive  kind  are  not  confined  to  the  interior  of  the  alimentary 
canal ;  but  most  of  the  body  cells  are  provided  with  ferments  to 
assist  them  either  in  utilising  the  nutrient  materials  brought  to  them 
by  the  blood-stream,  or  in  breaking  them  down  previously  to  expel- 
ling them  as  waste  substances.  The  ferment  which  enables  the  liver 
cells  to  turn  glycogen  into  sugar  is  the  one  which  has  been  known 
longest.  The  ferment  called  arginase  (see  p.  150),  which  leads  to 
the  hydrolysis  of  arginine  into  urea  and  ornithine,  is  one  of  the  most 
recently  discovered.  Other  examples  which  may  be  mentioned  are 
proteolytic  enzymes  (tissue  erepsin,  &c.)  found  in  many  organs. 

The  formation  of  uric  acid  from  nuclein  is  perhaps  the  best 
instance  of  all,  for  here  we  have  to  deal  with  numerous  ferments 
acting  one  after  another.  The  first  to  come  into  play  is  called 
nuclease ;  this  liberates  purine  bases  such  as  adenine  and  gua- 
nine from  the  nuclein ;  the  next  ferments  that  act  remove  the 
amino-group  from  the  purine  bases  just  mentioned ;  in  this  way 
adenine  (G5H3N4.NH0)  is  converted  into  hypoxanthine  C5H4N4O ; 
and  guanine  (C5H3N4O.NH2)  into  xanthine  C5H4N4O2.  These  two 
ferments  are  respectively  called  adenase  and  guanase.  Finally,  oxi- 
dising ferments,  or  oxidases,  step  in  and  oxidise  hypoxanthine  into 
xanthine,  and  xanthine  into  uric  acid.  By  examining  extracts  of 
various  organs,  the  distribution  of  these  numerous  ferments  has  been 
determined,  and  in  general  terms  the  spleen  and  liver  are  the  organs 
where  they  are  most  abundant.  But  the  examination  of  such  extracts 
has  shown  in  addition  that  the  list  of  ferments  is  not  yet  complete  ; 
for  some  extracts  in  part  break  up  the  uric  acid,  which  is  formed  into 
simpler  substances ;  the  uric  acid  destroying  ferment  is  called  the 
uricolytic  ferment.  We  therefore  learn  that  the  uric  acid  discharged 
in  the  urine  is  only  the  balance  left  over  when  the  amount  destroyed 
is  deducted  from  the  amount  originally  formed.  In  other  words,  the 
body  possesses  to  some  extent  the  power  of  protecting  itself  from  an 
excessive  formation  of  uric  acid,  and  so  from  the  evils  which  would 
result  from  an  accumulation  of  this  substance. 


162  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

HIPPURIC    ACID 

Hippuric  acid  (C9H9NO3),  combined  with  bases  to  form  hip- 
purates,  is  present  in  small  quantities  in  human  urine,  but  in  large 
quantities  in  that  of  herbivora.  This  is  due  to  the  food  of  herbivora 
containing  substances  belonging  to  the  aromatic  group — the  benzoic 
acid  series.  If  benzoic  acid  is  given  to  a  man,  it  unites  with  glycine 
with  the  elimination  of  a  molecule  of  water,  and  is  excreted  as 
hippuric  acid — 

CH2NH2        CH2NH.CO.C6H5 
CeHsCOOH  +  |  =  |  +H2O 

COOH  COOH 

[benzoic  acid]  [glycine]  [hippuric  acid]  [water] 

This  is  a  well-marked  instance  of  synthesis  carried  out  in  the 
animal  body,  and  experimental  investigation  shows  that  it  is  accom- 
plished by  the  living  cells  of  the  kidney  itself ;  for  if  a  mixture  of 
glycine,  benzoic  acid,  and  defibrinated  blood  is  injected  through  the 
kidney  (or  mixed  with  a  minced  kidney  just  removed  from  the  body  of 
an  animal),  their  place  is  found  to  have  been  taken  by  hippuric  acid. 

It  may  be  crystallised  from  horse's  urine  by  evaporating  to  a  syriip  and 
saturating  with  HCl.  The  crystals  are  dissolved  in  boiling  water,  filtered, 
and  on  cooling  the  acid  again  crystallises  out.  It  melts  at  186°  C,  and  on 
further  heating  gives  rise  to  the  odour  of  bitter  almond  oil. 

URINARY  DEPOSITS 

The  different  substances  that  may  occur  in  urinary  deposits  are 
formed  elements  and  chemical  substances. 

The  formed  or  histological  elements  may  consist  of  blood 
corpuscles,  pus,  mucus,  epithelium  cells,  spermatozoa,  casts  of  the 
urinary  tubules,  fungi,  and  entozoa.  All  of  these,  with  the  exception 
of  a  small  quantity  of  mucus,  which  forms  a  flocculent  cloud  in  the 
urine,  are  pathological,  and  the  microscope  is  chiefly  employed  in 
their  detection. 

The  chemical  substances  are  uric  acid,  urates,  calcium  oxalate, 
calcium  carbonate,  and  phosphates.  Earer  forms  are  leucine,  tyro- 
sine, xanthine,  and  cystin.  We  shall,  however,  here  only  consider 
the  commoner  deposits,  and  for  their  identification  the  microscope 
and  chemical  tests  must  both  be  employed. 

Deposit  of  Uric  Acid. — This  is  a  sandy  reddish  deposit  resembling 
cayenne  pepper.  It  may  be  recognised  by  its  crystalline  form  (fig. 
46,  p.  157)  and  by  the  murexide  reaction.  The  presence  of  these 
crystals  generally  indicates  an  increased  formation  of  uric  acid,  and, 


URINE  163 

if  excessive,  may  lead  to  the  formation  of  stones  or  calculi  in  the 
bladder. 

Deposit  of  Urates. — This  is  much  commoner,  and  may,  if  the 
urine  is  concentrated,  occur  in  normal  urine  when  it  cools.  It  is 
generally  found  in  the  concentrated  urine  of  fevers ;  and  there 
appears  to  be  a  kind  of  fermentation,  called  the  acid  fermentation, 
which  occurs  in  the  urine  after  it  has  been  passed,  and  which  leads 
to  the  same  result.  The  chief  constituent  of  the  deposit  is  the  acid 
sodium  urate,  the  formation  of  which  from  the  normal  sodium  urate 
of  the  urine  may  be  represented  by  the  equation — 

205H2Na,N4O3  +  H2O  +  C02=2C5H3NaN403  +  NagCOa 

[normal  sodium  [water]  [carbonic  [acid  sodium  [sodium 

urate]  acid]  urate]  carbonate] 

This  deposit  may  be  recognised  as  follows  : — 

1.  It  has  a  pinkish  colour  ;  the  pigment  called  uro-erythrin  is  one 


Fig.  47. — Acid  sodium  urate.  FiG.  48. — Acid  ammonium  urate. 

of  the  pigments  of  the  urine,  but  its  relationship  to  the  other  urinary 
pigments  is  not  known  (see  further  Lesson  XXVI.). 

2.  It  dissolves  upon  warming  the  urine. 

Microscopically  it  is  usually  amorphous,  but  crystalline  forms 
similar  to  those  depicted  in  figs.  47  and  48  may  occur. 

Crystals  of  calcium  oxalate  may  be  mixed  with  this  deposit  (see 
fig.  49). 

Deposit  of  Calcium  Oxalate. — This  occurs  in  envelope  crystals 
(octahedra)  or  dumb-bells. 

It  is  insoluble  in  ammonia,  and  in  acetic  acid.  It  is  soluble  with 
difficulty  in  hydrochloric  acid. 

Deposit  of  Cystin.—Gystin  (OgHisNiSaOi)  is  recognised  by  its 
colourless  six-sided  crystals  (fig.  50).  These  are  rare :  they  occur 
only  in  acid  urine,  and  they  may  form  concretions  or  calculi.  Cystin- 
uria  (cystin  in  the  urine)  is  hereditary. 

Deposit   of  Phosphates. — These   occur   in   alkaline    urine.     The 

M  2 


164 


ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 


urine  may  be  alkaline  when  passed,  due  to  fermentative  changes 
occurring  in  the  bladder.  All  urine,  however,  if  exposed  to  the  air 
(unless  the  air  is  perfectly  pure,  as  on  the  top  of  a  snow  mountain), 
will  in  time  become  alkaline  owing  to  the  growth  of  the  micrococciis 
itrecB.     This  forms  ammonium  carbonate  from  the  urea. 

[urea]  [water]         [ammonium 

carbonate] 

The  ammonia   renders  the  urine  alkaline,    and  precipitates  the 


^ 


0 

0    o 


Fiu.  49.— Envelope  crjstiils 
of  calcium  oxalate. 


Fig.  50.— Oystin  crystals. 


earthy  phosphates.     The   chief  forms  of  phosphates  that  occur  in 
urinary  deposits  are — 

1.  Calcium  phosphate,  Ca3(P04)2 ;  amorphous. 

2.  Triple  or  ammonio-magnesium  phosphate,  MgNH4P04  +  6H2O  ; 
coffin-hds  and  feathery  stars  (fig.  51). 


Fio.  51.— Triple  phosphate  crystals. 


Fjg.  52.— Crystals  of  phosphate  of  lime 
(stellar  phosphate). 


3.  Crystalline    phosphate   of   calcium,    CaHPO},    in   rosettes   of 
prisms,  in  spherules,  or  in  dumb-bells  (fig.  52). 

4.  Magnesium  phosphate,  Mg;3(P04).2  i  2H2O,  occurs  occasionally, 
and  crystallises  in  long  plates. 


UEINE 


165 


All  these  phosphates  are  dissolved  by  acids,  such  as  acetic  acid, 
without  effervescence. 

They  do  not  dissolve  on  heating  the  urine;  in  fact,  the  amount 
of  precipitate  may  be  increased  by  heating. 

A  solution  of  ammonium  carbonate  (l-in-5)  eats  magnesium 
phosphate  away  from  the  edges ;  it  has  no  effect  on  the  triple  phos- 
phate. A  phosphate  of  calcium  (CaHP04  +  2H20)  may  occasionally 
be  deposited  in  acid  urine.  Pus  in  urine  is  apt  to  be  mistaken  for 
phosphates,  but  can  be  distinguished  by  the  microscope. 

Deposit  of  calcium  carbonate,  CaCOa,  appears  but  rarely  as 
whitish  balls  or  biscuit-shaped  bodies.  It  is  commoner  in  the  urine 
of  herbivora  (see  p.  154).  It  dissolves  in  acetic  or  hydrochloric  acid, 
with  effervescence. 

The  following  is  a  summary  of  the  chemical  sediments  that  may 
occur  in  urine  : — 

CHEMICAL   SEDIMENTS   IN    UEINE 


In  Acid  Urine 

Uric  Acid. — Whetstone,  dumb- 
bell, or  sheaf-like  aggregations  of 
crystals  deeply  tinged  by  pigment 
(fig.  46). 

Urates.  —  Generally  amorphous. 
The  acid  urate  of  sodium  (fig.  47)  and 
of  ammonium  (fig.  48)  may  some- 
times occur  in  star-shaped  chisters  of 
needles  or  spheroidal  clumps  with 
projecting  spines.  Tinged  brick-red. 
Soluble  on  warming. 

Calcmm  Oxalate. —  Octahedra, 
so-called  envelope  crystals  (fig.  49). 
Insoluble  in  acetic  acid. 

Cystin. — Hexagonal  "plates  (fig. 
50).     Bare. 

Leucine  and  Tyrosine. — Kare. 

Calcium  Phosphate. 

CaHP04-f2H,0.— Rare. 


In  Alkaline  Urine 

Phosphates. — Calcium  phosphate, 
Ca3(P04)2.     Amorphous. 

Triple  phosphate, 
MgNH,P0,  +  6H,0.      Coffin-lids    or 
feathery  stars  (figs.  45  and  51). 

Calcium  hydrogen  phosphate, 
CaHPO^.  Rosettes,  spherules,  or 
dumb-bells  (fig.  52). 

Magnesium  phosphate, 
Mg3(P04).;j  +  2H.,0.     Long  plates. 

All  soluble  in  acetic  acid  without 
effervescence. 

Calciinn  Carbonate,  CaCO^. — 
Biscuit-shaped  crystals.  Soluble  in 
acetic  acid  with  effervescence. 

Ammonium  Urate, 
C,H,(NH,)o.N,03.  —  '  Thorn-apple  ' 
spherules. 

Leucine  and  Tyrosine. — Very  rare. 


LESSON   XII 
PATHOLOGICAL    UBINE 

1.  Urine  A  is  pathological  urine  containing  albumin.  It  gives  the  usual 
protein  tests.     The  following  two  are  most  frequently  used  in  practice. 

(rt)  Boil  the  top  of  a  long  column  of  urine  in  a  test-tube.  If  the  urine  is 
acid,  the  albumin  is  coagulated.  If  the  quantity  of  albumin  is  small,  the 
cloudiness  produced  is  readily  seen,  as  the  unboiled  urine  below  it  is  clear. 
This  is  insoluble  in  a  few  drops  of  acetic  acid,  and  so  may  be  distinguished 
from  phosphates.  If  the  urine  is  alkaline,  it  should  be  first  rendered  acid 
with  a  little  dilute  acetic  acid. 

(b)  Heller's  Nitric-Acid  Test. — Pour  some  of  the  urine  gently  on  to  the 
surface  of  some  nitric  acid  in  a  test-tube.  A  ring  of  white  precipitate  occurs 
at  the  junction  of  the  two  liquids.  This  test  is  used  for  small  quantities  of 
albumin. 

If  the  urine  is  cloudy,  it  should  be  filtered  before  applying  these  tests. 

2.  Estimation  of  Albumin  by  Esbach's  Albuminometer. — Esbach's  reagent 
for  precipitating  the  albumin  is  made  by  dissolving  10  grammes  of  picric  acid 
and  20  grammes  of  citric  acid  in  800  or  900  c.c.  of  boiling  water,  and  then 
adding  sufficient  water  to  make  up  to  a  litre  (1,000  c.c). 


:ir«i:?<i»:4i=<n»>'ifc 


FfG.  53. — Albuminometer  of  Esbach. 

Pour  the  urine  into  the  tube  up  to  the  mark  U  ;  then  the  reagent  up  to 
the  mark  R.  Close  the  tube  with  a  cork,  and  to  ensure  complete  mixture 
tilt  it  to  and  fro  a  dozen  times  without  shaking.  Allow  the  corked  tube  to 
stand  upright  twenty-four  hours  ;  then  read  off  on  the  scale  the  height  of  the 
precipitate.  The  figures  indicate  grammes  of  dried  albumin  in  a  litre  of  urine. 
The  percentage  is  obtained  by  dividing  by  10.  Thus,  if  the  sediment  stands 
at  3,  the  amount  of  albumin  is  3  grammes  per  litre,  or  0-3  gr.  in  100  c.c.  If 
the  sediment  falls  between  any  two  figures,  the  distance  },  i,  or  ^  from  the 
upper  or  lower  figure  can  be  read  off  with  sufficient  accuracy.  Thus  the 
surface  of  the  sediment,  being  midway  between  3  and  4,  would  be  read  as  3*5. 
When  the  albumin  is  so  abundant  that  the  sediment  is  above  4,  a  more 
accurate  result  is  obtained  by  first  diluting  the  urine  with  one  or  two  volumes 
of  water,  and  then  multiplying  the  resulting  figure  by  2  or  3,  as  the  case  may 
be.  If  the  amount  of  albumin  is  less  than  0*05  per  cent.,  it  cannot  be  accu- 
rately estimated  by  this  method. 

3.  Urine  B  is  diabetic  urine.  It  has  a  high  specific  gravity.  The  presence 
of  sugar  is  shown  by  the  reduction  (yellow  precipitate  of  cuprous  oxide)  that 
occurs  on  boiling  with  Fehling's  solution.  Fehling's  solution  is  an  alkaline 
solution  of  copper  sulphate  to  which  Rochelle  salt  has  been  added.  The 
Rochelle  salt  (double  tartrate  of  potash  and  soda)  holds  the  cupric  hydrate  in 


PATHOLOGICAL  URINE 


167 


solution.  Fehling's  solution  should  always  be  freshly  prepared,  as,  on  stand- 
ing, an  isomeride  is  formed  from  the  tartaric  acid,  and  this  substance  itself 
reduces  the  cupric  to  cuprous  oxide.  Fehling's  solution  should,  therefore, 
always  be  tested  by  boiling  before  it  is  used.  If  it  remains  unaltered  by 
boiling,  it  is  in  good  condition. 

4.  Quantitative  Determination  of  Sugar  in  Urine. — Fehling's  solution  is  pre- 
pared as  follows  : — 34*639  grammes  of  copper  sulphate  are  dissolved  in  about 
200  c.c.  of  distilled  water ;  173  grammes  of  Eochelle 
salt  are  dissolved  in  600  c.c.  of  a  14-per-cent.  solution 
of  caustic  soda.  The  two  solutions  are  mixed  and 
diluted  to  a  litre.  Ten  c.c.  of  this  solution  are 
equivalent  to  0*05  gramme  of  dextrose.  Dilute  10  c.c. 
of  this  solution  with  about  40  c.c.  of  water,  and  boil  it 

■in  a  white  porcelain  dish.  Run  into  this  from  the 
burette  (see  fig.  54)  the  virine  (which  should  be 
previously  diluted  with  nine  times  its  volume  of 
distilled  water)  until  the  blue  colour  of  the  copper 
solution  disappears — that  is,  till  all  the  cupric  hj^drate 
is  reduced.  The  mixture  in  the  basin  should  be  boiled 
after  every  addition.^  The  quantity  of  diluted  urine 
used  from  the  burette  contains  O'Oo  gramme  of  sugar. 
Calculate  the  percentage  from  this,  remembering  that 
the  urine  has  been  diluted  to  ten  times  its  original 
volume. 

Example.— Su-pTpose  that  20  c.c.  of  the  diluted 
urine  are  found  necessary  to  reduce  the  10  c.c.  of 
Fehling's  solution.  This  wiU  be  equivalent  to  2  c.c. 
of  the  undiluted  urine  ;  2  c.c.  of  the  original  urine 
will  therefore  contain  0*05  gramme  of  sugar ;  1  c.c. 

will  contain    ^^  and  100  c.c.  will  contain     ^^  x  100 
2  2 

=  2'5  grammes  of  sugar. 

Pavy's  modification  of  Fehling's  solution  is  some- 
times used.  Here  ammonia  holds  the  cuprous  oxide 
in  solution,  so  that  no  precipitate  forms  on  boiling 
Pavy's  solution  with  a  reducing  sugar.  The  reduc- 
tion is  complete  when  the  blue  colour  disappears :  10  c.c.  of  Pavy' 
tion  =  1  c.c.  of  Fehling's  solution  =  0*005  gramme  of  dextrose. 

In  some  cases  of  diabetic  urine  where  there  is  excess  of  ammonio- 
magnesic  phosphate,  the' full  reduction  is  not  obtained  with  Fehling's  solu- 
tion, and  when  the  quantity  of  sugar  is  small  it  may  be  missed.  In  such  a 
case  excess  of  soda  or  potash  should  be  first  added ;  the  precipitated 
phosphates  filtered  off;  and  the  filtrate,  after  it  has  been  well  boiled,  may 
then  be  titrated  with  Fehling's  or  Pavy's  solution. 

5.  Picric  Acid  Test.—  The  work  of  Sir  George  Johnson  and  G.  S.  Johnson 
has  shown  the  value  of  this  reagent  in  detecting  both  albumin  and  sugar 
in  the  urine.  The  same  reagent  may  be  employed  for  the  detection  of 
both  substances.  The  method  of  testing  for  albumin  has  been  already  studied 
with  Esbach's  tubes.  To  test  for  sugar  perform  the  following  experiment. 
Take  a  drachm  (about  4  c.c.)  of  diabetic  urine  ;  add  to  it  an  equal  volume  of 
saturated  aqueous  solution  of  picric  acid,  and  half  the  volume  {i.e.  2  c.c.)  of 
the  liquor  potassae  of  the  British  Pharmacopoeia.  Boil  the  mixture  for  about 
a  minute,  and  it  becomes  so  intensely  dark  red  as  to  be  opaque.  Now  do 
the  same  experiment  with  normal  urine.  An  orange-red  colour  appears 
even  in  the  cold,  and  is  deepened  by  boiling,  but  it  never  becomes  opaque, 


I'Ki.  54. — Two  burettes  on 
stand.    (Sutton.) 


solu- 


On  cooling  the  blue  colour  reappears,  owing  to  re-oxidation. 


168  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

and  so  the  urine  for  clinical  purposes  may  be  considered  free  from  sugar. 
This  reduction  of  picric  acid  by  normal  urine  is  due  to  creatinine. 

6.  Test  for  Acetone. — Acetone  is  often  found  in  diabetic  urine.  Add  to  the 
urine  a  dilute  solution  of  sodium  nitro-prusside  and  a  little  20-per-cent. 
caustic  potash.  A  red  colour  is  produced.  Acidify  with  strong  acetic 
acid.  The  colour  disappears  at  once  in  the  absence  of  acetone,  but  re- 
mains or  is  intensified  in  its  presence. 


The  full  significance  and  causes  of  pathological  urine  cannot  be 
appreciated  until  a  theoretical  and  practical  acquaintance  with  disease 
is  obtained,  and  we  shall  briefly  consider  only  those  abnormal  con- 
stituents which  are  most  frequently  met  with. 

PROTEINS    IN   THE   URINE 

There  is  no  protein  matter  in  normal  urine,  and  the  most  common 
cause  of  the  appearance  of  albumin  in  the  urine  is  disease  of  the 
kidney  (Bright's  disease).  The  best  methods  of  testing  for  and  esti- 
mating the  albumin  are  given  in  the  practical  heading  to  this  lesson. 
The  term  *  albumin '  is  the  one  used  by  clinical  observers.  Properly 
speaking,  it  is  a  mixture  of  serum  albumin  and  serum  globulin. 

A  condition  called  'peptonuria,'  or  peptone  in  the  urine,  is  ob- 
served in  certain  pathological  states,  especially  in  diseases  where 
there  is  a  formation  of  pus,  and  particularly  if  the  pus  is  decomposing 
owing  to  the  action  of  a  bacterial  growth  called  staphylococcus ;  one 
of  the  products  of  disintegration  of  pus  cells  appears  to  be  peptone ; 
and  this  leaves  the  body  by  the  urine.  The  term  '  peptone,'  however, 
is  in  the  strict  sense  incorrect  ;  the  protein  present  is  deutero- 
proteose.  In  the  disease  called  *  osteomalacia  '  a  proteose  is  usually 
found  in  the  urine,  which  more  nearly  resembles  hetero-proteose  in 
its  characters. 

SUGAR   IN   THE  URINE 

Normal  urine  contains  no  sugar,  or  so  little  that  for  clinical  pur- 
poses it  may  be  considered  absent.  It  occurs  in  the  disease  called 
diabetes  mellitus,  which  can  be  artificially  produced  by  the  methods 
described  on  pp.  87,  88. 

The  methods  usually  adopted  for  detecting  and  estimating  the 
sugar  are  given  at  the  head  of  this  lesson.  The  sugar  present  is 
dextrose.  Lactose  may  occur  in  the  urine  of  nursing  mothers. 
Diabetic  urine  also  contains  hydroxybutyric  acid,  and  may  contain 
or  yield  on  distillation  acetone  and  ethyl  diacetic  acid. 

Fehling's  test  is  not  absolutely  trustworthy.  Often  a  normal  urine  will 
decolorise  Fehling's  solution,  though  seldom  a  red  precipitate  is  formed. 
This  is  due  to  excess  of  urates  and  creatinine.     Another  substance,  called 


PATHOLOGICAL  UEINE  169 

glycuronic  acid  (C^HjqO-)  is,  however,  very  likely  to  be  confused  with 
sugar  by  Fehling's  test ;  the  cause  of  its  appearance  is  sometimes  the 
administration  of  drugs  (chloral,  camphor,  &c.) ;  but  sometimes  it  appears 
independently  of  drug  treatment  (see  p.  88). 

Then,  too,  in  the  rare  condition  called  alcaptonuria,  confusion  may 
similarly  arise.  Alcapton  is  a  substance  which  probably  originates,  from 
tyrosine  by  an  unusual  form  of  metabolism.  It  gives  the  urine  a  brown  tint, 
which  darkens  on  exposure  to  the  air.  It  is  an  aromatic  substance,  and  the 
researches  of  Baumann  and  Wolkow  have  identified  it  with  homogentisinic 
acid  [C,H3.(0H),CH,.C00H]. 

The  best  confirmatory  tests  for  sugar  are  the  phenyl-hydrazine 
test  (see  Lesson  XIII.),  and  i\\Q  fermentation  test  (Lesson  IL). 

Sir  W.  Eoberts  introduced  a  method  for  estimating  sugar  in  urine,  by  the 
diminution  in  specific  gravity  which  it  undergoes  on  fermentation  with  yeast. 
Ever}-  degree  lost  in  the  specific  gravity  corresponds  to  one  grain  of  sugar  per 
fluid  ounce.  Suppose  that  the  specific  gravity  of  the  unfermented  urine  is 
1040,  and  that  of  the  urine  which  has  undergone  fermentation  is  1030 :  the 
number  of  degrees  lost  is  ten  ;  i.e.  the  urine  contained  10  grains  of  sugar  per 
ounce.  The  percentage  of  sugar  may  be  ascertained  by  multiplying  the 
degrees  of  specific  gravity  lost  by  0*22 ;  thus  the  percentage  in  the  example 
just  given  will  be  0*22  x  10  =  2*2.  The  method,  however,  is  rough  and  has 
dropped  out  of  use. 

BILE  IN   THE   URINE 

This  occurs  in  jaundice.  The  urine  is  dark-brown,  greenish,  or 
in  extreme  cases  almost  black  in  colour.  The  most  readily  applied 
test  is  Gmelin's  test  for  the  bile  pigments.  Pettenkofer's  test  for  the 
bile  acids  seldom  succeeds  in  urine  if  the  test  is  done  in  the  ordinary 
way.  The  best  method  is  to  warm  a  thin  film  of  urine  and  cane- 
sugar  solution  in  a  flat  porcelain  dish.  Then  dip  a  glass  rod  in 
strong  sulphuric  acid,  and  draw  it  across  the  film.  Its  track  is 
marked  by  a  purplish  line.  Hay's  sulphur  test  (p.  79)  is  very  trust- 
worthy.    Excess  of  urobilin  should  not  be  mistaken  for  bile  pigment. 

BLOOD   AND  BLOOD   PIGMENT   IN   THE  URINE 

When  haemorrhage  occurs  in  any  part  of  the  urinary  tract,  blood 
appears  in  the  urine.  It  is  found  in  the  acute  stage  of  Bright's 
disease.  If  a  large  quantity  is  present,  the  urine  is  deep  red. 
Microscopic  examination  then  reveals  the  presence  of  blood  cor- 
puscles, and  on  spectroscopic  examination  the  bands  of  oxyhaemo- 
globin  are  seen. 

If  only  a  small  quantity  of  blood  is  present,  the  secretion — 
especially  if  acid — has  a  characteristic  reddish-brown  colour,  which 
physicians  term  *  smoky.' 

The  blood  pigment  may,  under  certain  circumstances,  appear  in 


170  ESSENTIALS   OF  CHEMICAL   PHYSIOLOGY 

the  urine  without  the  presence  of  any  blood  corpuscles  at  all.  This 
is  produced  by  a  disintegration  of  the  corpuscles  occurring  in  the 
circulation.  The  condition  so  produced  is  called  hcBmoglobiiiuria, 
and  it  occurs  in  several  pathological  states,  as,  for  instance,  in  the 
tropical  disease  known  as  '  Black-water  fever.'  The  pigment  is  in 
the  condition  of  methaemoglobin  mixed  with  more  or  less  oxyhaemo- 
globin,  and  the  spectroscope  is  the  means  used  for  identifying  these 
substances  (see  p.  119). 

PUS  IN  THE   URINE 

Pus  occurs  in  the  urine  as  the  result  of  suppuration  in  any  part 
of  the  urinary  tract.  It  forms  a  white  sediment  resembling  that  of 
phosphates,  and,  indeed,  is  always  mixed  with  phosphates.  The  pus 
corpuscles  may,  however,  be  seen  with  the  microscope ;  their  nuclei 
are  rendered  evident  by  treatment  with  1-per-cent.  acetic  acid,  and 
the  pus  corpuscles  are  seen  to  resemble  white  blood  corpuscles, 
which,  in  fact,  they  are  in  origin.  Some  of  the  protein  constituents 
of  the  pus  cells — and  the  same  is  true  for  blood — pass  into  solution, 
so  that  the  urine  pipetted  off  from  the  surface  of  the  deposit  gives 
the  tests  for  albumin.  On  the  addition  of  liquor  potassae  to  the 
deposit  of  pus  cells  a  ropy  gelatinous  mass  is  obtained.  This  is 
distinctive.     Mucus  treated  in  the  same  way  is  dissolved. 


DETECTION   OF   PHYSIOLOGICAL    PROXIMATE    PRINCIPLES 

Subsequent  lessons  may  be  very  usefully  employed  by  the  class  in  testing 
for  the  various  substances  the  properties  of  which  have  been  previously 
studied.  The  following  scheme  will  form  a  rough  guide  to  the  tests  to  be 
employed  for  the  most  important  of  the  proximate  principles  : — 

1.  Note  reaction,  colour,  clearness  or  opalescence,  taste,  smell.  Coloured 
liquids  suggest  blood,  bile,  urine,  &c.  Opalescent  liquids  suggest  starch, 
glycogen,  or  certain  proteins, 

2.  Add  iodine.     A  colour  is  produced  : 

If  blue  :  Starch.  Confirm  by  converting  into  a  reducing  sugar  by  saHva 
at  40°  C,  or  by  boiling  with  dilute  sulphuric  acid. 

If  reddish  brown :  Glycogen  or  dextrin.  Glycogen  forms  an  opalescent 
solution  in  water,  and  is  readily  precipitated  by  alcohol.  It  is  precipitated 
by  basic  lead  acetate.  Dextrin  forms  a  clear  solution  :  it  is  not  precipitated 
by  basic  lead  acetate  unless  ammonia  is  added  also.  It  is  not  precipitated  by 
alcohol  unless  a  large  excess  is  added.  Both  dextrin  and  glj'cogen  are,  like 
starch,  convertible  into  a  reducing  sugar. 

3.  Add  copper  sulphate  and  caustic  potash. 

(«)  Blue  solution :  boil ;  yellow  or  red  precipitate.  Dextrose,  levulose, 
maltose,  lactose,  and  other  reducing  sugars  (for  distinguishing  tests  see 
Lesson  XIII.). 

(b)  Blue  solution :  no  reduction  on  boiling ;  boil  some  of  the  original 
solution  with  25-per-cent.  sulphuric  acid,  and  then  boil  with  copper  sulphate 
and  caustic  potash ;  abundant  yellow  or  red  precipitate  :  Cane  sugar.  Confinn 
by  HCl  test  (see  p.  13). 

(c)  Violet  solution  :  Proteins  (albumins,  globulins,  infra-proteins).  In 
presence  of  magnesium  sulphate  the  potash  causes  also  a  white  precipitate  of 
magnesia. 

(d)  Pink  solution ;  biuret  reaction.  Peptones  or  proteoses.  In  presence 
of  ammonium  sulphate  very  large  excess  of  potash  is  necessary  for  this  test. 
Only  a  trace  of  copper  sulphate  must  be  used. 

4.  "When  proteins  are  present  proceed  as  follows  :  Boil  the  original  solution 
(after  adding  a  trace  of  2-per-cent.  acetic  acid). 

(a)  Precipitate  produced  :  Albumins  or  globulins. 

(b)  No  precipitate  :  Infra-proteins,^  proteoses,  or  peptones. 

5.  If  albumin,  or  globulin,  or  both  are  present,  saturate  a  fresli  portion 
with  magnesium  sulphate  or  half  saturate  with  ammonium  sulphate;  filter; 
the  precipitate  contains  the  globulin,  the  filtrate  the  albumin.  Test  tempera- 
ture of  heat  coagulation. 

'  The  infra-proteins  (see  pp.  47  and  75)  are  by  some  called  '  meta-proteins.' 


172  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

6.  If  proteins  are  present,  but  albumin  or  globulin  absent : 

(a)  Neutralisation  causes  a  precipitate  soluble  in  excess  of  weak  acid  or 
alkali.  Acid  albumin  or  alkali  albumin,  according  as  the  reaction  of  the 
original  liquid  is  acid  or  alkaline  respectively.  If  the  original  liquid  is 
neutral,  acid  albumin  and  alkali  albumin  must  be  both  absent. 

(6)  Neutralisation  produces  no  such  precipitate  :  Proteose  or  peptone. 

7.  If  proteose,  or  peptone,  or  both  are  present,  saturate  a  fresh  portion 
with  ammonium  sulphate : 

(rt)  Precipitate  :  Proteose.     (&)  No  precipitate  :  Peptone. 
If  both  are  present,  the  precipitate  contains  the  proteose,  and  the  filtrate 
the  peptone. 

8.  To  a  fresh  portion  add  nitric  acid  (proteins  having  been  proved  to  be 
present). 

(a)  No  precipitate,  even  though  excess  of  sodium  chloride  be  also  added : 
Peptone. 

(6)  No  precipitate,  until  excess  of  sodium  chloride  is  added :  Deutero- 
proteose. 

(c)  Precipitate  which  disappears  on  heating  and  reappears  on  cooling : 
Proteoses.  This  is  the  distinctive  test  of  all  the  proteoses  or  albumoses,  and 
is  given  by  all  of  them.  For  one  of  them,  however  (deutero-proteose),  excess 
of  sodium  chloride  must  be  added  also. 

(d)  Precipitate  little  altered  by  heating:  Albumin  or  globulin. 

In  all  four  cases  nitric  acid^jZi^s  heat  causes  a  yellow  colour,  tinned  orange 
by  ammonia  (xantho-proteic  reaction). 

9.  Confirmatory  tests  for  proteins : — 
{a)  Millon's  test  (see  p.  27). 

(6)  Adamkiewicz's  reaction  (see  p.  27). 

(c)  Ferrocyanide  of  potassium  and  acetic  acid  cause  a  precipitate  (except 
in  the  case  of  peptones  and  some  proteoses). 

(d)  To  test  for  fibrinogen : — 

i.  It  coagulates  by  heat  at  56°  C. 

ii.  It  is  changed  into  fibrin  by  fibrin  ferment  and  calcium  chloride. 

(e)  To  test  for  caseinogen  : — 
1.  It  is  not  coagulated  by  heat. 

ii.  It  is  changed  into  casein  by  rennet  and  calcium  chloride. 

10.  If  blood  is  suspected  : 

(a)  Examine  spectroscopically,  diluting  if  necessary. 

i.  Oxyhaemoglobin  shows  two  bands  between  D  and  E. 

ii.  Add  ammonium  sulphide  ;  one  band  only  appears. 

iii.  Carbonic  oxide  haemoglobin  shows  two  bands  also,  but  will  not  reduce 
with  ammonium  sulphide. 

iv.  Methsemoglobin  gives  a  typical  band  in  the  red  between  C  and  D. 

v.  Haematin,  &c.,  show  special  spectra  (see  Advanced  Course,  Lesson  XIX.). 

(6)  Dry :  boil  with  glacial  acetic  acid  and  a  crystal  of  sodium  chloride  on 
a  glass  slide  under  a  cover  glass.     When  cold,  haemin  crystals  are  seen. 


DETECTION  OF  PROXIMATE   PRINCIPLES  173 

(c)  If  the  blood  is  old  and  dry,  and  its  haemoglobin  converted  into 
hsematin : 

i.  Try  haemin  test. 

ii.  Dissolve  it  in  potash  ;  add  ammonium  sulphide,  and  examine  for 
spectrum  of  haemochromogen. 

11.  If  bile  is  suspected  : 

(a)  Try  Gmelin's  test  for  bile  pigments  (see  p.  79). 
(6)  Try  Pettenkofer's  test  for  bile  salts  (see  p.  79). 
(c)  Try  Hay's  sulphur  test  (see  p.  79). 

12.  Miscellaneous  substances. 

(a)  Mucin.  Precipitated  by  acetic  acid  or  by  alcohol.  The  precipitate  is 
soluble  in  lime  water.  By  collecting  the  precipitate  and  boiling  it  with  25- 
per-cent.  sulphuric  acid,  a  reducing  sugar-like  substance  is  obtained.  Mucin 
gives  the  protein  colour  tests. 

(b)  Nucleo-protein. — Precipitated  by  acetic  acid  or  by  alcohol.  The  pre- 
cipitate is  often  viscous.  It  is  soluble  in  dilute  alkalis  such  as  1-per-cent. 
sodium  carbonate.  This  solution  causes  intravascular  clotting.  If  the  pre- 
cipitate is  collected  and  subjected  to  gastric  digestion,  an  insoluble  deposit  of 
nuclein  is  left,  which  is  rich  in  phosphorus.  Nucleo-protein  gives  the  protein 
colour  tests. 

(c)  Gelatin.  This  also  gives  some  of  the  proteid  colour  tests,  but  not 
those  of  Millon  or  Adamkiewicz.  It  is  not  coagulated,  but  dissolved  in  hot 
water.     The  solution  gelatinises  when  cold. 

(d)  Urea.  Very  soluble  in  water.  The  solution  effervesces  when  sodium 
hypobromite  or  fuming  nitric  acid  is  added.  Concentrate  a  fresh  portion, 
add  nitric  acid,  and  examine  for  crystals  of  urea  nitrate.  Solid  urea  heated 
in  a  dry  test-tube  gives  off  ammonia,  and  the  residue  is  called  biuret.  This 
gives  a  rose-red  colour  with  copper  sulphate  and  caustic  potash. 

(e)  Uric  acid.  Very  insoluble  in  water ;  soluble  in  potash,  and  precipitated 
from  this  solution  in  crystals  by  hydrochloric  acid.  Uric  acid  crystals  from 
human  urine  are  deeply  pigmented  red.     Try  murexide  test  (see  p.  156). 

(/)  Cholesterin.  Characteristic  flat  crystalline  plates.  For  various  colour 
tests  see  p.  79. 

13.  Urine.     Normal  constituents 

(a)  Chlorides.  Acidulate  with  nitric  acid ;  add  silver  nitrate ;  white 
precipitate. 

(6)  Sulphates.  Acidulate  with  nitric  or  hj'drochloric  acid  :  add  barium 
chloride ;  white  precipitate. 

(c)  Phosphates.  Acidulate  with  nitric  acid  ;  add  ammonium  molybdate  ; 
boil;  and  a  yellow  crystalline  precipitate  forms.  To  another  portion  add 
ammonia ;  earthy  {i.e.  calcium  and  magnesium)  phosphates  are  precipitated. 

(d)  Urea  (see  above). 

(e)  Uric  acid.  To  100  c.c.  of  urine  add  5  c.c.  of  hydrochloric  acid ;  leave 
for  twenty-four  hours,  and  pigmented  crystals  of  uric  acid  are  formed.  For 
tests  see  above. 

(/)  Hippuric  acid.  Evaporate  the  urine  with  nitric  acid,  and  heat  the 
residue  in  a  dry  test-tube.     A  smell  of  oil  of  bitter  almonds  is  given  off. 


174  ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 

(g)  Creatinine.  Take  100  c.c.  of  urine  :  add  5  c.c.  of  a  saturated  solution 
of  sodium  acetate  and  20  c.c.  of  a  saturated  solution  of  mercuric  chloride- 
Filter.  Set  the  filtrate  aside  for  twenty-four  hours,  and  a  spherical  mercury 
compound  of  creatinine  crystallises  out.     Examine  this  with  a  microscope. 

For  colour  test  with  sodium  nitro-prusside  see  p.  142. 

14.  TTrine.     Abnormal  constituents. 

{a)  Blood.  Microscope  (blood  corpuscles).  Spectroscope  (for  oxyhaemo- 
globin  or  methaemoglobin).     Haemin  test. 

(6)  Blood  pigment  may  be  present  without  blood  corpuscles.  Spectro- 
scope. 

(c)  Bile.     Gmelin's  test.     Hay's  sulphur  test. 

(d)  Pus.  White  deposit.  Microscope  (pus  cells).  Add  potash  ;  it  becomes 
stringy. 

{e)  Albumin,  (i.)  Precipitated,  if  acid,  by  boiling ;  precipitate  insoluble 
in  acetic  acid,  so  distinguishing  it  from  phosphates,  (ii.)  Precipitated  by 
nitric  acid  in  the  cold,     (iii.)  Precipitated  by  picric  acid. 

(/)  Sugar,  (i.)  Brown  colour  with  potash  and  heat  (Moore's  test),  (ii.) 
Ferments  with  yeast,  (iii.)  Keduces  Fehling's  solution,  (iv.)  Urine  has  a 
high  specific  gravity,  (v.)  Add  picric  acid,  potash,  and  boil ;  the  urine 
becomes  a  dark  opaque  red ;  the  similar  slight  coloration  in  normal  urine  is 
due  to  creatinine. 

(jjl)  Acetone.     For  colour  test  see  p.  168. 

(7i)  Mucus.  Flocculent  cloud  ;  may  be  increased  by  acetic  acid  ;  soluble 
in  alkalis.     A  little  mucus  in  urine  is  not  abnormal. 

(i)  Deposits. 

i.  Examine  microscopically  for  blood  corpuscles,  pus  cells,  crj^stals,  &c. 

ii.  Phosphates,  White  deposit  often  mixed  with  nmcus  or  pus.  Insoluble 
on  heating ;  soluble  in  acetic  acid.  Urine  generally  alkaline.  Examine 
microscopically  for  coffin-lids  of  triple  phosphate  and  star-like  clusters  of 
stellar  (calcium)  phosphate. 

iii.  Urates.  Pink  deposit,  usually  amorphous;  may  be  mixed  with 
envelope  crystals  of  calcium  oxalate.  Deposit  soluble  on  heating  urine. 
Murexide  test. 

iv.  Uric  acid.  Deposit  like  cayenne  pepper.  Microscope.  Tests  as 
above. 


ADVANCED    COUESE 


INTKODUCTION 

It  will  be  presupposed  that  students  who  take  the  following  lessons  have 
already  been  through  the  elementary  course.  The  order  in  which  the 
subjects  are  treated  is  the  same  as  that  already  adopted.  The  instructions 
given  will  be  mainly  practical ;  theoretical  matter  on  which  they  depend, 
or  to  which  they  lead,  is,  as  a  rule,  too  lengthy  to  be  discussed  in  a  short 
manual  like  the  present  volume.  The  Appendix  contains  a  description  of 
various  instruments  which  are  not  generally  contained  in  sufficient  numbers 
in  a  physiological  laboratory  to  admit  of  each  student  being  able  to  use  them 
in  a  class.  It  also  contains  a  description  of  certain  methods  of  research 
which  should  always  be  shown  in  demonstrations,  though  there  may  be 
practical  difficulties  in  allowing  each  member  of  the  class  to  perform  the 
experiments.  The  few  experiments  in  which  living  animals  are  employed 
will  also  necessarily  be  of  the  nature  of  demonstrations. 


LESSON   XIII 

CARBOHYDRATES 


1.  Glycogen. — A  rabbit  which  has  been  fed  five  or  six  hours  previously 
on  carrots  is  killed  by  bleeding.  The  chest  and  abdomen  are  opened  quickly 
and  a  cannula  inserted  into  the  portal  vein,  and  another  into  the  vena  cava 
inferior.  A  stream  of  salt  solution  is  then  allowed  to  pass  through  the  liver 
until  it  is  uniformly  pale.  The  washings  are  collected  in  three  beakers 
labelled  a,  b,  and  c. 

The  liver  is  cut  out  quickly,  chopped  into  small  pieces,  and  thrown  into 

boiling  water  acidulated 
with  acetic  acid.  The 
acidulated  water  extracts 
a  small  quantity  of  glyco- 
gen. The  pieces  of  scalded 
liver  are  then  ground  up 
in  a  mortar  with  hot 
water,  and  thoroughly 
extracted  with  boiling 
water.  Filter.  A  strong 
solution  of  glycogen  is 
thus  obtained. 

Test  the  solution  when 
cold  with  iodine. 

To  separate  the  glyco- 
gen evaporate  the  solu- 
tion to  a  small  bulk  on  the 
water-bath  and  then  add 
excess  of  alcohol  ;  the 
glycogen  is  precipitated 
as    a  flocculent    powder, 

which  is  collected  on  a  filter  and  dried  in  an  oven  at  the  temperature  of  100° 

(see  fig.  55). 

If  the  experiment  is  to  be  a  quantitative  one^  the  piece  of  liver  taken  and 

the  glycogen  obtained  must  be  weighed.^ 

2.  Examine  the  washings  of  the  liver  in  the  beakers  a,  b,  and  c  for  sugar. 
This  may  be  done  in  a  rough  quantitative  manner  as  follows : — Take  equal 

'  This  method  of  preparation  of  glycogen  has  the  advantage  that  only  traces 
of  protein  are  mixed  with  it.  In  Kiilz's  method  (extraction  with  dilute  potash) 
there  is  more  protein.  This  is  precipitated  by  the  alternate  addition  of  hydro- 
chloric  acid  and  potassio-mercuric  iodide.  Pavy  and  also  Pfliiger  recommend 
extraction  with  strong  potash,  and  subsequent  precipitation  with  a  certain  per- 
centage of  alcohol ;  this  method  extracts  all  the  glycogen  easily. 


Fig.  55. — Hut-air  oven  with  gas  reguUitor  (<;).    (Gscheidlen.) 


Fig.  56.— Plate  of  osazone  crystals  highly  magnified. 
A,  phmyl-glucosazone.     B,  phenyl-maUosazone.     C\  phenyl-lactosazone. 


CARBOHYDRATES  177 

quantities  of  a,  b,  and  c  in  three  test-tubes ;  to  each  add  an  equal  amount  of 
FehHng's  sohition,  and  boil :  a  will  give  a  heavy  precipitate  of  cuprous 
oxide,  b  one  not  so  heavy,  and  c  least  of  all,  or  none  at  all. 

3.  Micro-chemical  detection  of  Glycogen. — A  thin  piece  of  the  same  liver  is 
hardened  in  90  per  cent,  alcohol.  Sections  are  cut  by  the  free  hand,  or  after 
embedding  in  paraffin.  If  paraffin  is  used,  this  is  got  rid  of  by  means  of 
turpentine;  and  the  sections  prepared  by  either  method  are  treated  with 
chloroform  m  which  iodine  is  dissolved,  and  mounted  in  chloroform  balsam 
containing  some  iodine.  The  glycogen  is  stained  brown,  and  is  most  abundant 
in  the  cells  around  the  radicals  of  the  hepatic  vein. 

4.  Phenyl-Hydrazine  Test  for  Sugars. — To  5  c.c.  of  the  suspected  fluid  {e.g. 
diabetic  urine)  add  1  decigramme  of  phenyl-hydrazine  hydrochloride,  2  deci- 
grammes of  sodium  acetate,  and  heat  on  the  water-bath  at  100*"  C.  for  30  to 
60  minutes.  On  cooling,  if  not  before,  a  crystalline  or  amorphous  precipitate 
separates  out.  If  amorphous,  dissolve  it  in  hot  alcohol ;  dilute  the  solution 
with  water,  and  boil  to  expel  the  alcohol,  whereupon  the  osazone  separates 
out  in  yellow  crystals.  Examine  the  crystals  with  the  microscope  (see 
accompanying  plate). 

Dextrose  gives  a  precipitate  of  phenyl-glucosazone  C,3Hio04(N2H.CgH3)2, 
which  crystallises  in  yellow  needles  (melting-point  205°  C). 

Levulose  yields  an  osazone  identical  with  this. 

Galactose  yields  a  very  similar  osazone  (phenyl-galactosazone).  It  differs 
from  phenyl-glucosazone  by  melting  at  190-198°,  and  in  being  optically  in- 
active when  dissolved  in  glacial  acetic  acid. 

Cane  sugar  does  not  form  a  compound  with  phenyl-hydrazine. 

Lactose  yields  phenyl-lactosazone  0,2H.3oOj)(NoH.CyIl5).^.  It  crystallises 
in  needles,  usually  in  clusters  (melting-point  200°  C).  It  is  soluble  in 
80-90  parts  of  boiling  water.  Lactose  in  urine  does  not  give  this  test 
readily. 

Maltose  yields  phenyl-maltosazone  (C2|H32N40y).  It  crystallises  in  yellow 
needles  much  wider  than  those  yielded  by  ghicose  or  lactose  (melting-point 
206^  C).  Unlike  phenyl-glucosazone,  it  dissolves  in  75  parts  of  boiling  water 
and  is  still  more  soluble  in  hot  alcohol. 

The  chemistry  of  the-  phenyl-hydrazine  reaction  is  represented  in  the 
following  equations,  dextrose  being  taken  as  an  example  of  the  sugar  used  :- 

I.  CH20H[CH(0H)]  3CH(0H)C0H  -f  H,N.NH(C6H5) 

[dextrose]  [phenyl-hydrazine] 

CH.0H[CH(0H)]3CH(0H)CH 

II  +        H,0 

N-NH(C6H,) 

[hydrazone]  .  [water] 


II.  CH20H[CH(OH)]3CH(OH)CH 


N  -  NH(CoH,) 

[hydrazone]  [phenyl-hydrazine] 

CH.OH  [CH(OH)]  3C  -  CH 

II     II  +     H2     -f     H2O 

CeH^.NH-N    N-NH.C,H, 

[osazone]  [hydrogen]        [water] 

N 


178  ESSENTIALS  OF  CHEMICAL  PHYSIOLOaY 


5.  Barfoed's  Reagent. — Dissolve  1  part  of  cupric  acetate  in  15  parts  of 
water ;  to  200  c.c.  of  this  solution  add  5  c.c.  of  acetic  acid  containing  38  per 
cent,  of  glacial  acetic  acid.  Dextrose  reduces  this  reagent  on  boiling ; 
maltose  and  lactose  do  not.  This  test  is  not  very  delicate,  and  the  reagent 
must  be  freshly  prepared. 

6.  The  Polarimeter. — Estimate  the  strength  of  a  solution  of  dextrose  by 
means  of  the  polarimeter  (see  Appendix). 

7.  Formation  of  Mucic  Acid. — Take  1  gramme  of  lactose  and  heat  it  in  a 
porcelain  capsule  with  12  c.c.  of  nitric  acid  on  a  water-bath  until  the  fluid  is 
reduced  to  one-third  of  its  original  volume.  A  precipitate  of  mucic  acid 
separates  out.  Cane  sugar,  maltose,  dextrose,  dextrin,  and  starch  treated  in 
the  same  way,  yield  an  isomeric  acid  called  saccharic  acid,  which,  being 
soluble,  does  not  separate  out.  Lactose  yields  both  acids ;  galactose  mucic 
acid  only. 

8.  Pentoses  give  the  ordinary  reduction  tests  for  sugar  and  yield  osazones, 
but  do  not  ferment  with  yeast.  They  give  the  two  following  characteristic 
tests ;  they  may  be  performed  with  gum  arable  (which  contains  arabinose) 
or  pine- wood  shavings  (which  contain  xylose). 

(a)  Phloroglucin  reaction.  Warm  some  distilled  water  with  an  equal 
vohime  of  concentrated  hydrochloric  acid  in  a  test-tube  and  add  phloroglucin 
until  a  little  remains  undissolved.  Add  a  small  quantity  of  gum  arable, 
and  keep  the  mixture  warm  in  the  water-bath  at  100°  C.  The  solution 
becomes  cherry-red,  and  a  precipitate  settles  out,  which  is  soluble  in  amyl 
alcohol.     This  solution  gives  an  absorption  band  between  the  D  and  E  lines. 

(b)  Orcin  reaction.  Substitute  orcin  for  phloroglucin  in  the  foregoing 
experiment.  The  solution  becomes  violet  on  warming,  then  blue,  red,  and 
finally  green.  A  bluish  green  precipitate  settles  out,  soluble  in  amyl  alcohol. 
This  solution  gives  an  absorption  band  between  C  and  D. 

9.  Glycuronic  acid  gives  all  the  above  reactions  ;  it  may  be  distinguished 
as  follows : — 

Take  50  c.c.  of  glycuronic  acid  solution  in  a  capsule  ;  add  1  gramme  of 
p-bromphenyl-hydrazine  and  rather  more  than  the  same  amount  of  sodium 
acetate.  Keep  the  mixture  in  the  water-bath  at  100°  C.  for  a  quarter  of  an 
hour,  when  yellow  crystals  of  ^;-bromphenyl-hydrazone  separate  out.  After 
cooling  filter  off  the  crystals  and  wash  them  with  absolute  alcohol,  in  which 
they  are  insoluble.  Under  the  same  conditions  carbohydrates  yield  ^j-brom- 
phenyl-osazones,  but  these  ^re  soluble  in  absolute  alcohol.  The  j)-brom- 
phenyl-hydrazone  is  soluble  in  absolute  alcohol  to  which  pyridine  has  been 
added ;  the  rotatory  power  of  this  solution  is  greater  than  that  of  any  of  the 
osazones. 


LESSON   XIV 
CARBOHYDRATES :  ACTION  OF  MALT    UPON  STARCH 

1.  Prepare  a  0'5-per-cent.  solution  of  starch. 

2.  Prepare  some  malt  extract  by  digesting  10  grammes  of  powdered  malt 
with  50  CO.  of  water  at  50°  C.  for  three  hours,  and  subsequently  straining. 
This  extract  contains  the  diastatic  or  malting  ferment. 

Solutions  1  and  2  may  be  conveniently  prepared  beforehand  by  the 
demonstrator. 

3.  To  the  starch  solution  add  one-tenth  of  its  volume  of  malt  extract,  and 
place  the  mixture  in  a  water-bath  at  40°  C.  From  time  to  time  test  portions 
of  the  liquid  by  mixing  a  drop  with  a  drop  of  iodine  solution  on  a  testing 
slab.  The  blue  colour  at  first  seen  is  soon  replaced  by  a  violet  (mixture  of 
blue  and  red),  and  then  by  a  red  reaction  (due  to  erythrodextrin),  which 
gradually  vanishes.  Alcohol  added  to  the  liquid  when  all  starch  and  erythro- 
dextrin  have  gone  still  causes  a  precipitate  of  a  dextrin,  which,  as  it  gives  no 
colour  with  iodine,  is  called  achron -dextrin.  The  liquid  also  contains  a 
reducing  sugar,  maltose. 

4.  Take  50  c.c.  of  a  solution  of  maltose  and  determine  how  much  of  it 
is  necessary  to  reduce  10  c.c.  of  Fehling's  solution. 

5.  Take  another  50  c.c.  and  boil  it  with  1  c.c.  of  strong  sulphuric  acid  fo 

half  an  hour  in  a  flask.     This  converts  it  into  dextrose.     After  cooling  bring 

the  liquid  to  its  original  volume  (50  c.c.)  by  adding  water,  and  again  determine 

its  increased  reducing  power  with  Fehling's  solution.     If  j?  =  c.c.  of  maltose 

2x 
solution  necessary  to  reduce  10  c.c.  of  Fehling's  solution,  then    ^    -  c.c.  of 

o 

dextrose  solution  necessary  for   the    same   purpose.     The    strength    of  the 

maltose  solution  can  be  calculated  from  the  fact  that  10  c.c.  of  Fehling's 

solution  correspond  to  0*05  gramme  of  dextrose. 


N  2 


LESSON   XV 
CBYSTALLISATION  OF  EGG   ALBUMIN 

Fresh  egg-^white  is  mixed  with  an  equal  bulk  of  fully  saturated,  filtered 
neutral  ammonium  sulphate  solution.  100  c.c.  of  the  former  are  measured 
into  a  porcelain  basin  or  strong  beaker,  and  100  c.c.  of  ammonium  sulphate 
solution  are  added  in  successive  quantities  of  10  or  15  c.c,  the  mixture  being 
thoroughly  churned  with  an  egg  whisk  after  each  addition.  The  whole  should 
be  finally  so  thoroughly  beaten  up  as  to  form  a  large  proportion  of  light  froth. 
After  the  greater  part  of  the  froth  has  broken  down,  the  mixture  is  thrown  on 
a  folded  filter-paper,  moderately  rapid  filtration  being  obtained  without  the 
use  of  a  filter-pump.  The  filtrate  is  strongly  alkaline  to  litmus,  and  smells  of 
ammonia.  To  the  filtrate  in  a  flask,  or  to  as  much  of  it  as  can  be  obtained  in 
a  convenient  time  of  filtration,  further  ammonium  sulphate  solution  is  very 
cautiously  added  (best,  drop  by  drop  from  a  burette)  until  a  slight  permanent 
precipitate  remains,  and  this  precipitate  is  afterwards  just  redissolved  by  the 
equally  cautious  addition  of  water.  Dilute  acetic  acid  (10  per  cent.)  from  a 
burette  is  now  added  drop  by  drop  until  such  a  stage  of  reaction  is  reached  that 
a  precipitate  forms  and  only  just  redissolves.  Finally  one  or  two  drops  (not 
more)  of  acid  are  added  in  excess  of  this,  whereupon  a  bulky  white  precipitate 
falls.  The  flask  is  now  corked  and  allowed  to  stand.  In  24  hours  or  less  the 
precipitate,  which  will  have  increased  in  quantity,  will  be  found  to  consist 
entirely  of  acicular  crystals.  Small  portions  should  be  examined  under  a 
^th  objective,  avoiding  pressure  on  the  cover  slip.     (F.  G.  Hopkins.) 


LESSON  XVI 

MILK 

1.  Caseinogen  in  milk  exists  in  the  form  of  a  salt  (calcium  caseinogenate). 
Add  acetic  acid  to  milk,  and  this  salt  is  decomposed,  and  free  caseinogen 
(with  entangled  fat)  is  precipitated.  Collect  the  precipitate  so  produced  from 
a  pint  of  milk  on  a  filter,  and  wash  thoroughly  with  distilled  water :  grind  it 
up  with  calcium  carbonate  in  a  mortar,  and  add  about  a  pint  of  distilled 
water ;  allow  the  mixture  to  stand  for  about  an  hour.  The  fat  rises  to  the 
top  :  the  excess  of  calcium  carbonate  falls  to  the  bottom.  The  intermediate 
fluid  contains  the  caseinogen  in  solution  ;  it  is  usually  very  opalescent. 
Take  some  of  this  solution  and  divide  it  into  three  parts,  A,  B,  and  C. 

To  A  add  rennet. 

To  B  add  a  few  drops  of  2-per-cent.  solution  of  calcium  chloride. 
To  C  add  both  rennet  and  calcium  chloride. 

Put  all  three  in  the  water-bath  at  40^  C.  A  clot  of  casein  forms  in  C,  but 
not  in  A  if  all  calcium  salts  have  been  successfully  washed  away,  nor  in  B. 

2.  The  formation  of  casein  from  caseinogen  is  a  double  process ;  the  first 
action  is  that  of  the  ferment,  which  converts  the  caseinogen  into  what  may 
be  called  soluble  casein ;  the  second  action  is  that  of  the  calcium  salt,  which 
precipitates  the  casein  in  an  insoluble  form,  or  curd.  This  may  be  shown  by 
taking  some  of  the  caseinogen  solution  and  adding  rennet.  Warm  to  40°  C. ; 
no  visible  change  occurs,  but  nevertheless  soluble  casein  and  not  caseinogen 
is  now  present.  Then  boil  this  mixture  to  destroy  the  rennet,  cool,  and  add 
calcium  chloride.     A  formation  of  insoluble  curd  now  occurs. 

3.  Caseinogen  may  be  precipitated  as  a  salt  from  milk  by  the  addition  of 
alcohol.     This  reagent  also  precipitates  the  other  milk  proteins. 

4.  The  method  of  salting  out  described  in  Lesson  VI.,  Exercise  10  (p.  41), 
may  also  be  used.  Add  to  some  milk  an  equal  volume  of  saturated  solution 
of  ammonium  sulphate..  Caseinogen  as  a  salt  is  thus  precipitated,  and 
entangles  the  fat;  with  it.  Filter  off  the  precipitate  and  examine  the  filtrate 
as  follows  : — Saturate  it  with  sodium  chloride  ;  a  small  amount  of  precipitate 
comes  down.  This  is  the  so-called  lacto-globulin.  This  contains  only  a 
trace  of  true  globulin ;  it  is  mostly  caseinogen  previously  left  in  solution 
together  with  calcium  sulphate.  Filter  it  off;  acidify  the  filtrate  with  a  few 
drops  of  2-per-cent.  acetic  acid  and  heat  it  in  a  water-bath  gradually.  About 
77°  C.  the  remaining  protein  (lactalbumin)  is  coagulated. 


LESSON  XVII 

THE  PROTEOSES 

1.  Witte's  peptone  contains  very  little  true  peptone,  but  consists  chiefly 
of  proteoses,  which  are  soluble  like  peptone,  in  neutral  saline  solutions. 

2.  Make  a  solution  of  this  substance  in  10-per-cent.  sodium  chloride 
solution,  and  filter.  Very  little  residue  is  left  on  the  filter.  This  consists  of 
dysalbumose,  an  insoluble  form  of  hetero-albumose,  formed  during  the  pro- 
cess of  preparing  the  substance.  If  hot  saline  solution  is  used  instead  of  cold 
as  a  solvent,  this  amount  of  insoluble  residue  is  increased,  hetero-albumose 
being  to  a  slight  extent  precipitated  by  heat. 

3.  The  solution  gives  the  following  tests : — 

(a)  It  does  not  coagulate  on  heating. 

(b)  Biuret  reaction  (due  both  to  peptone  and  proteoses). 

(c)  A  drop  of  nitric  acid,  best  added  hj  a  glass  rod,  gives  a  precipitate 
which  dissolves  upon  heating  and  reappears  on  cooling.  (This  is  due  to  the 
proteoses  present.) 

(d)  The  precipitate  produced  by  the  addition  of  acetic  acid  and  a  drop  of 
potassium  ferrocyanide  is  also  soluble  on  heating,  and  reappears  on  cooling. 

4.  For  the  separation  of  the  proteoses  and  peptone  proceed  as  follows : — 

(a)  Saturate  the  solution  with  ammonium  sulphate,  and  filter.  The 
filtrate  contains  the  peptone,  and  the  precipitate  the  proteoses.  The  peptone 
is  not  precipitated  by  nitric  acid,  nor  by  most  of  the  reagents  that  precipitate 
other  proteins.  It  is  precipitated  completely  by  alcohol,  tannin,  and  potassio- 
mercuric  iodide  ;  imperfectly  by  phospho-tungstic  and  phospho-molybdic  acid. 

It  gives  the  biuret  reaction,  bid  in  the  jJresence  of  aminoniiim  sulphate  a 
large  excess  of  caustic  potash  is  necessary. 

(b)  Dialyse  another  portion  of  the  solution ;  hetero-proteose  is  pre- 
cipitated. 

(c)  Saturate  another  portion  of  the  solution  with  sodium  chloride  (or  half 
saturate  with  ammonium  sulphate)  after  faintly  acidulating  with  acetic  acid. 
Proto-proteose  and  hetero-proteose  are  precipitated.  Filter.  The  filtrate 
contains  the  deutero-proteose  and  peptone. 

The  proto-  and  hetero-proteose  may  be  redissolved  by  adding  distilled 
water,  and  may  be  separated  from  each  other  by  dialysis  (see  b). 

Deutero-proteose  may  be  separated  from  the  peptone  by  saturation  with 
ammonium  sulphate,  or  by  the  addition  of  a  crystal  of  phosphoric  acid. 
These  reagents  precipitate  the  deutero-proteose,  but  not  the  peptone. 

Deutero-proteose  gives  the  nitric  acid  reaction  (see  3,  c)  characteristic  of 
the  proteoses  only  in  the  presence  of  excess  of  salt.  If  the  salt  is  removed 
by  dialysis,  nitric  acid  then  causes  no  precipitate. 


THE   PROTEOSES 


183 


5.  Among  the  important  reactions  of  proteins  is  Rose's  or  Piotrowski's 
reaction — that  is,  the  coloration  produced  by  copper  sulphate  and  a  caustic 
alkali ;  the  term  '  biuret  reaction  '  is  applied  to  the  rose-red  colour  which 
proteoses  and  peptones  give  with  these  reagents,  because  biuret  (a  derivative 
of  urea)  gives  a  similar  colour  (see  p.  39).  Gnezda  found  that  if  a  dilute 
solution  of  nickel  sulphate  is  used  instead  of  copper  sulphate,  the  native  pro- 
teins give  different  colours  from  the  peptones  and  proteoses,  and  Pickering 
has  found  the  same  with  cobalt.  Their  results  may  be  given  in  the  following 
table  : — 


Proteiu 

Copper  sul-    Copper  sul- 
phate and       phate  and 
ammonia           potash 

Nickel  sul- 
phate and 
ammonia 

Nil 

Nickel  sul- 
phate and 
potash 

Cobalt  sul- 
phate and 
ammonia 

Cobalt  sul- 
phate and 
potash 

Albumins  and\ 
globulins      / 

Blue        ':       Violet 

Yellow 
Orange 

Nil 

Heliotrope- 
purple      1 

Proteoses  and  \ 
peptones        / 

Rose-red         Rose-red 

i 

Yellow 

Nil 

Red  browu 

6.  Another  delicate  test  introduced  by  McWilliam  may  here  be  mentioned. 
Salicyl-sulphonic  acid  precipitates  albumins  and  globulins  :  on  heating,  the 
precipitate  is  coagulated.  The  same  reagent  precipitates  proteoses.  On 
heating,  the  precipitate  dissolves  and  reappears  on  cooling.  It  does  not 
precipitate  peptones. 

7.  The  use  of  trichloracetic  acid  for  the  separation  of  various  proteins 
may  be  illustrated  by  the  following  experiment.  Take  some  blood  and  add 
to  it  some  solution  of  Witte's  peptone  {i.e.  proteoses  and  peptone).  Add  to 
this  mixture  an  equal  volume  of  a  10-per-cent.  solution  of  trichloracetic  acid. 
There  is  an  abundant  precipitate.  Boil  rapidly  and  filter  hot.  The  filtrate 
contains  the  proteoses  and  peptone,  all  the  other  proteins  being  contained  in 
the  precipitate.  On  cooling,  the  filtrate  deposits  some  of  the  proteose.  The 
proteose  and  peptone  may  be  detected  in  the  usual  way. 


LESSON   XVIII 

DIGESTION 

1.  Activity  of  Pepsin  Solutions  (Griitzner's  Method). — Examine  the  com- 
parative digestive  power  of  the  glycerin  extracts  of  two  stomachs.  Take,  in 
two  test-tubes,  an  equal  small  weighed  quantity  of  fibrin  stained  with  carmine. 
Add  to  each  10  c.c.  of  0'2-per-cent.  hydrochloric  acid.  Add  to  one  a  measured 
quantity  of  one  glycerin  extract,  and  to  the  other  an  equal  quantity  of  the 
other  glycerin  extract.  As  the  fibrin  is  digested  the  carmine  is  set  free,  and 
colours  the  liquid ;  that  which  is  more  deeply  stained  is  that  which  contains 
the  more  active  preparation  of  pepsin.  •  In  the  original  method  the  amount 
of  carmine  set  free  is  estimated  by  an  artificial  scale  consisting  of  ten  solutions 
of  carmine  of  different  known  strengths. 

The  carmine  solution  for  staining  the  fibrin  is  prepared  by  dissolving 
1  gramme  of  carmine  in  about  1  c.c.  of  ammonia  ;  to  this  400  c.c.  of  water 
are  added,  and  the  mixture  is  kept  in  a  loosely  stoppered  bottle  till  the  smell 
of  ammonia  has  become  faint. 

The  fibrin  is  stained  by  taking  it  perfectly  fresh  and  clean.  It  is  chopped 
finely  and  placed  in  the  carmine  solution  for  twenty-four  hours.  The  fluid  is 
strained  off  and  the  fibrin  washed  in  water  till  the  washings  are  colourless. 
It  is  kept  in  a  stoppered  bottle  with  just  enough  ether  to  cover  it. 

2.  Mett's  Tubes.— A  method  which  is  now  more  generally  employed  for 
estimating  the  proteolytic  activity  of  a  digestive  juice  is  one  originally 
introduced  by  Mett.  Pieces  of  capillary  glass  tubing  of  known  length  are 
filled  with  white  of  egg.  This  is  set  into  a  solid  by  heating  to  95°  C.  They 
are  then  placed  in  the  digestive  fluid  at  36°  C,  and  the  coagulated  egg-white 
is  digested.  After  a  given  time  the  tubes  are  removed ;  and  if  the  digestive 
process  has  not  gone  too  far,  onh'  a  part  of  the  little  column  of  coagulated 
protein  will  have  disappeared ;  the  length  of  the  remaining  column  is  easily 
measured,  and  the  length  that  has  been  digested  is  a  measure  of  the  digestive 
strength  of  the  fluid.'  This  forms  a  very  convenient  method  to  use  in  experi- 
ments on  velocity  of  reaction.  Schiitz's  Law  states  that  the  amount  of  action 
is  proportional  to  the  square  root  of  the  amount  of  ferment.  If  this  rule 
applies  (which  is  doubtful)  it  applies  only  to  the  action  of  pepsin  hydrochloric 

'  Hamburger  has  used  the  same  method  in  investigating  the  digestive  action  of 
juices  on  gelatin.  The  tubes  are  filled  with  warm  gelatin  solution,  and  this  jellies 
on  cooling.  They  are  placed  as  before  in  the  digestive  mixture,  and  the  length  of 
the  column  that  disappears  can  be  easily  measured.  These  experiments  must,  how- 
ever, be  performed  at  room  temperature,  for  the  usual  temperature  (.36°-40°  C.)  at 
which  artificial  digestion  is  usually  carried  out  would  melt  the  gelatin.  He  has  also 
used  the  same  method  for  estimating  araylolytie  activity,  by  filling  the  tubes  with 
thick  starch  paste. 


DIGESTION  186 

acid.  In  most  cases  the  rapidity  of  action  »is  directly  proportional  to  the 
amount  of  ferment  present. 

3.  The  Acid  of  Gastric  Juice. — The  digestive  powers  of  the  acids  are  pro- 
portional to  their  dissociation  and  the  number  of  H  ions  liberated.  The 
anions,  however,  modify  this  by  having  different  powers  of  retarding  the 
action.  The  greater  suitability  of  hydrochloric  over  lactic  acid,  for  instance, 
in  gastric  digestion  is  due  to  the  fact  that  the  former  acid  more  readily 
undergoes  dissociation. 

Hydrochloric  acid  is  absent  or  diminished  in  some  diseases  of  the 
stomach,  especially  in  cancer  ;  this  is  true  for  cancer  in  general  even  when 
the  stomach  is  not  involved ;  the  best  colour  tests  for  it  are  the  following : — 

(a)  Gunsberg's  reagent  consists  of  2  parts  of  phloroglucinol,  1  part  of 
vanillin,  and  30  parts  of  rectified  spirit.  A  drop  of  filtered  gastric  juice  is 
evaporated  with  an  equal  quantity  of  the  reagent.  Ked  crystals  form,  or,  if 
much  peptone  is  present,  there  will  be  a  red  paste.  The  reaction  takes  place 
with  one  part  of  hydrochloric  acid  in  10,000.  The  organic  acids  do  not  give 
the  reaction. 

(6)  Tropaeolin  test.  Drops  of  a  saturated  solution  of  tropseolin-00  in 
94  per  cent,  methylated  spirit  are  allowed  to  dry  on  a  porcelain  slab  at  40°  C. 
A  drop  of  the  fluid  to  be  tested  is  placed  on  the  tropaeolin  drop,  still  at  40°  C. ; 
and  if  hydrochloric  acid  is  present  a  violet  spot  is  left  when  the  fluid  has 
evaporated.  A  di'op  of  0'006-per-cent.  hydrochloric  acid  leaves  a  distinct 
mark.  , 

(c)  Topfer's  test.  A  drop  of  dimethyl-amino-azo-benzol  is  spread  in  a 
thin  film  on  a  white  plate.  A  drop  of  dilute  hydrochloric  acid  (up  to  1  in 
10,000)  strikes  with  this  in  the  cold  a  bright  red  colour. 

Lactic  acid  is  sometimes  present  in  the  gastric  contents,  being  derived  by 
fermentative  processes  from  the  food.  It  is  soluble  in  ether,  and  is  generally 
detected  by  making  an  ethereal  extract  of  the  stomach  contents,  and  evapo- 
rating the  ether.  If  lactic  acid  is  present  in  the  residue  it  may  be  identified 
by  Uffelmann's  reaction  in  the  following  way  : — 

A  solution  of  dilute  ferric  chloride  and  carbolic  acid  is  made  as  follows  :  - 

10  c.c.  of  a  4-per-ceut.  solution  of  carbolic  acid. 

20  c.c.  of  distilled  water. 

1  drop  of  the  liquor  ferri  perchloridi  of  the  British  Pharmacopoeia. 

On  mixing  a  solution  containing  a  mere  trace  (up  to  1  part  in  10,000)  of 
lactic  acid  with  this  violet  solution,  it  is  instantly  turned  yellow.  Larger 
percentages  of  other  acids— for  instance,  more  than  0*2  per  cent,  of  hydro- 
chloric acid — are  necessary  to  decolorise  the  test  solution. 

The  reaction  is  not  absolutely  convincing,  since  other  acids  (though  in 
larger  percentages)  decolorise  the  solution,  but  the  characteristic  yellow  colour 
given  even  by  dilute  lactic  acid  is  not  developed.  Note  the  decolorisation 
which  occurs  when  0*2  hydrochloric  acid  is  added  to  Uffelmann's  reagent. 

Hopkins's  reaction  for  Lactic  Acid. — Place  3  drops  of  a  1-per-cent.  alco- 
holic solution  of  lactic  acid  in  a  clean,  dry  test-tube,  add  5  c.c.  of  concen- 
trated sulphuric  acid  and  3  drops  of  a  saturated  solution  of  copper  sulphate. 
Mix  thoroughly  and  place  the  test-tube  in  a  beaker  of  boiling  water  for  five 
minutes.    Then  cool  thoroughly  under  the  tap,  and  add  2  drops  of  a  0*2-per- 


186  ESSENTIALS  OF  CHEMICAI.  PHYSIOLOGY 

cent,  alcoholic  solution  of  thiophene  and  shake.     Replace  the  tube  in  the 
boiling  water ;  as  the  mixture  gets  warm  a  cherry-red  colour  develops. 

4.  Demonstration  of  Pancreatic  Secretion. — In  an  anaesthetised  dog  insert 
a  cannula  into  the  main  pancreatic  duct,  and  collect  the  juice  in  a  suitable 
vessel.  Inject  some  0-4-per-cent.  hydrochloric  acid  into  the  duodenum,  and 
note  after  some  minutes  the  abundant  flow  of  pancreatic  juice.  Next 
ligature  off  and  remove  two  or  three  feet  of  the  upper  part  of  the  small 
intestine,  wash  out  the  contents  and  slit  it  open  ;  scrape  off  the  mucous 
membrane  with  the  back  of  a  scalpel ;  preserve  a  small  quantity  of  the 
scrapings  for  future  use  and  label  this  A.  Grind  up  the  remainder  in  a 
mortar  with  clean  sand  or  powdered  glass,  and  add  0*4  per  cent,  hydrochloric 
acid.  Transfer  the  mixture  to  a  flask,  boil  thoroughly,  and  when  cool 
neutralise  with  a  little  caustic  soda  solution.  Filter ;  the  filtrate  contains 
secretin,  which  has  been  formed  by  the  acid  from  the  ^jro-secretin  of  the 
intestinal  epithelium.  Inject  some  of  this  solution  through  a  cannula  into 
the  external  jugular  vein  of  the  dog,  and  an  abundant  flow  of  pancreatic 
juice  is  an  almjst  immediate  result. 

5.  Characters  of  the  juice  so  obtained : — 

{a)  It  is  a  clear,  colourless  fluid,  and  very  strongly  alkaline. 

(6)  Mixed  with  starch  solution  and  kept  at  40°  C.  dextrin  and  maltose  are 
rapidly  formed. 

(c)  Mixed  with  milk  there  may  be  some  curdling  produced,  but  the  most 
marked  effect  is  that  the  milk  rapidly  becomes  acid,  and  a  smell  of  fatty 
acids  is  noticeable. 

{d)  Added  to  fibrin  and  kept  at  40°  protein  digestion  occurs  very  slowly; 
next  day,  however,  the  fibrin  will  be  in  large  measure  digested. 

{e)  Mix  some  of  the  pancreatic  juice  with  the  scraping  of  the  intestine 
which  was  preserved  and  labelled  A.  Then  add  fibrin.  The  fluid  is  now 
strongly  proteolytic,  and  at  40°  C.  the  fibrin  rapidly  dissolves  ;  trypsin  has 
been  liberated  from  the  trypsinogen  of  the  juice  by  the  intestinal  entero- 
Tiinase. 

6.  Products  of  Pancreatic  Digestion  of  Proteins. — A  pancreatic  digest 
should  be  prepared  beforehand  by  the  demonstrator.  This  may  be  done  by 
digesting  a  quantity  of  protein  with  artificial  pancreatic  juice,  if  the  natural 
juice  prepared  by  the  action  of  secretin  is  not  available ;  in  the  latter  case 
the  addition  of  intestinal  epithelium  (entero-kinase)  should  not  be  forgotten. 
Unless  an  antiseptic  has  been  added  putrefaction  will  also  occur,  and  its 
odour  will  be  very  perceptible  after  the  mixture  has  been  placed  in  the  warm 
chamber  for  some  time. 

A  very  good  mixture  for  the  purpose  will  be  found  to  be  the  following  :— 

100  grammes  of  plasmon  (caseinogen). 
10  grammes  of  sodium  carbonate. 

1  litre  of  water. 
25  c.c.  of  Benger's  liquor  pancreaticus. 

0*5  gramme  sodium  fluoride. 

3  c.c.  chloroform. 

The  last  two  items  on  the  list  are  added  to  prevent  putrefaction. 


i)iaESTioN  187 

After  digestion  has  progressed  for  one  to  two  days  another  10  c.c.  of  Hquor 
pancreaticus  may  be  added. 

The  products  of  digestion  in  one  case  should  be  examined,  say,  after  six 
hours'  digestion,  and  in  another  case  after  thirty-six  hours'  digestion  or  more. 
The  digestive  products  should  then  be  searched  for  ;  the  early  products  of 
digestion  (alkali-albumin,  deutero-proteose,  &c.)  will  become  less  abundant 
with  the  length  of  time  that  digestion  has  been  allowed  to  progress,  and  the 
later  products  (peptone,  leucine,  tyrosine,  tryptophane,  &c.)  will  become  more 
abundant.  The  methods  for  testing  most  of  these  substances  have  been 
already  given.  The  following  are  the  tests  for  tryptophane,  leucine,  and 
tyrosine  : — 

(a)  Try2}to;plian6. — Add  a  few  drops  of  bromine  water  ;  a  violet  colour  is 
produced. 

(6)  Leucine  and  Ty7'osine. — i.  Examine  microscopical  specimens  of  these. 
The  deposit  generally  found  in  rather  old  specimens  of  Banger's  liquor  pan- 
creaticus will  be  a  convenient  source  of  these  crystals. 

ii.  To  some  of  the  pancreatic  digest  add  Millon's  reagent  and  filter  ofi' 
the  precipitated  protein.  Boil  the  filtrate,  and  the  presence  of  tyrosine  is 
indicated  by  a  red  colour.  If  tyrosine  is  abundant  the  red  colour  appears 
without  boiling.     Leucine  does  not  give  this  test. 

iii.  Faintly  acidify  another  portion  of  the  filtered  digest  with  acetic  acid 
and  boil ;  if  any  protein  matter  is  still  undigested  it  will  be  thus  coagulated 
and  can  be  filtered  off.  Eeduce  the  filtrate  to  a  small  bulk  until  it  begins  to 
become  syrupy.  Leave  overnight  in  a  cool  place,  and  crystals  mainly  of 
tyrosine  will  separate  out.  Filter  these  off  through  fine  muslin,  and 
evaporate  down  the  filtrate  to  the  consistency  of  a  thick  syrup ;  leave  this 
overnight  again,  and  a  second  crop  of  crystals,  forming  a  skin  on  the  surface 
and  consisting  mainly  of  leucine,  will  have  separated  out. 

7.  Zymogen  Granules. — Examine  microscopically,  mounting  in  aqueous 
humour  or  serum  (or  in  glycerin  after  treatment  with  osmic  acid  vapour), 
small  pieces  of  the  pancreas,  parotid,  and  submaxillary  glands  in  a  normal 
guinea-pig,^  and  also  in  one  in  which  profuse  secretion  had  been  produced  by 
the  administration  of  pilocarpine. 

Note  that  zymogen  granules  are  abundant  in  the  former,  and  scarce  in 
the  latter,  being  situated  chiefly  at  the  free  border  of  the  cells. 

Extremely  good,  though  not  permanent,  microscopic  specimens  may  be 
obtained  by  teasing  in  a  33-per-cent.  solution  of  caustic  potash. 

'  The  guinea-pigs  should  be  killed  by  bleeding,  and  the  blood  collected  and 
defibrinated,  and  utilised  for  the  preparation  of  oxyheemoglobin  crystals.  This 
will  give  students  an  opportunity  of  seeing  the  exceptional  form  (tetrahedra)  in 
which  the  blood-pigment  of  this  animal  crystallises. 

The  three  methods  of  obtaining  crystals  described  on  p.  112  all  give  good  results. 
If  amyl  nitrite  is  used  instead  of  ether  in  the  third  method,  crystals  of  methasmo- 
globin  are  obtained. 


LESSON   XIX 

HEMOGLOBIN  AND  ITS  DERIVATIVES 

Defibrinated  ox-blood  suitably  diluted  may  be  used  in  the  following 
experiments  as  in  those  described  in  Lesson  IX. 

1.  Place  some  in  a  haematoscope  (see  fig.  33,  p.  116)  in  front  of  the  large 
spectroscope.  Note  the  position  of  the  two  characteristic  bands  of  oxyhaemo- 
globin ;  these  are  replaced  by  the  single  band  of  hsemoglobin  after  reduction  by 
the  addition  of  Stokes's  reagent  (see  footnote,  p.  115)  or  ammonium  sulphide. 
By  means  of  a  small  rectangular  prism  a  comparison  spectrum  showing 
the  bright  sodium  line  (in  the  position  of  the  dark  line  named  D  in  the  solar 
spectrum)  may  be  obtained,  and  focussed  with  the  absorption  spectrum. 

2.  Obtain  similar  comparison  spectra  by  the  use  of  the  microspectroscope. 
For  this  purpose  a  cell  containing  a  small  quantity  of  oxyhaemoglobin 
solution  may  be  placed  on  the  microscope  stage,  and  a  test-tube  containing 
carbonic  oxide  hgemoglobin  in  front  of  the  slit  in  the  side  of  the  instrument. 
Notice  that  the  two  bands  of  carbonic  oxide  hsemoglobin  are  very  like  those 
of  oxyhaemoglobin,  but  are  a  little  nearer  to  the  violet  end  of  the  spectrum. 

Carbonic  oxide  haemoglobin  may  be  readily  prepared  by  passing  a  stream 
of  coal  gas  through  the  diluted  blood.  It  has  a  cherry-red  colour  and  is  not 
reduced  by  the  addition  of  ammonium  sulphide  (fig.  57,  spectrum  4). 

3.  Methsemoglobin. — Add  a  few  drops  of  ferricyanide  of  potassium  to 
dilute  blood  and  warm  gently.     The  colour  changes   to  mahogany-brown. 

•Place  the  test-tube  in  front  of  the  small  direct-vision  spectroscope.  Note 
the  characteristic  band  in  the  red  (fig.  57,  spectrum  5).  On  dilution  other 
bands  appear  (fig.  57,  spectrum  6).  Treat  with  ammonium  sulphide  and 
the  band  of  haemoglobin  appears. 

4.  Acid  Haematin. — (a)  Prepare  the  following  mixture  : — 150  c.c.  of  90-per- 
cent, alcohol  and  6  c.c.  of  concentrated  sulphuric  acid ;  take  about  5  c.c.  of 
this  mixture  and  boil  it  in  a  test-tube.  While  still  hot  drop  into  it  a  few 
drops  of  undiluted  defibrinated  blood,  and  filter.  Note  the  brown  colour  of 
the  filtrate.  Compare  the  position  of  the  absorption  band  in  the  red  with 
that  of  methaemoglobin  ;  that  of  acid  haematin  is  further  from  the  D  line 
(fig.  57,  spectrum  7). 

(b)  Add  some  glacial  acetic  acid  to  undiluted  defibrinated  blood.  Extract 
this  with  ether  by  gently  agitating  it  with  that  fluid.  The  ethereal  extract 
should  then  be  poured  off  and  examined  spectroscopically.  The  band  in  the 
red  is  seen,  and  on  further  diluting  with  ether  three  additional  bands  appear. 

5.  Alkaline  Haematin. — {a)  Add  to  diluted  blood  a  small  quantity  of  strong 


HEMOGLOBIN 


189 


Fio.  57.— 1,  Solar  spectrum.  2,  Spectrum  of  oxyhEemoglobin  (0*37  p.c.  solution).  First  band,  A  589- 
564  :  second  band,  A  555-517.  3,  Spectrum  of  haemoglobin.  Band,  A  597-535.  4.  Spect-um  of  CO- 
haemoglobin.  First  band,  A  583-56  i  ;  second  band.  A  547-521.  5,  Spectrum  of  methsemoglobin 
(concentrated  solution).  6,  Speatrum  of  methaemoglobin  (dilute  solution).  First  band,  A  647- 
622:  second  band,  A  587-571;  tliird  band,  A  552-532;  fourth  band,  A  514-490.  7,  Spectrum  of 
acid  lisematin  (ethereal  solution).  First  band,  A  656-615  ;  second  band,  A  597-577  ;  third  band,  A 
557-529;  fourth  band,  A  517-488.  8,  Spectrum  of  alkaline  haematin.  Band  from  A  630-581. 
9,  Spectrum  of  hfemochromogen  (reduced  haBmatin).  First  band,  A  569-542 ;  second  band,  A 
535-504.  10,  Spectriim  of  acid  htematoporphyrin.  First  band,  A  607-593  ;  second  band,  A  585- 
536.  11,  Spectrum  of  alkaline  hajmatoporphyrin.  First  baud,  A  633-612  ;  second  band,  A  589-564 ; 
third  band,  A  549-529  ;  fourth  band,  A  518-488.  The  above  measurements  (after  MacMunu)  are 
in  millionths  of  a  millimetre.  The  1  quid  was  examined  in  a  layer  1  centimetre  thick.  The  edges 
of  ill-defined  bands  vary  a  good  deal  with  Dhe  concentration  of  the  solutions. 


190 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


caustic  potash  and  boil.  The  colour  changes  to  brown,  and  with  the 
spectroscope  a  faint  shading  on  the  left  side  of  the  D  line  is  seen  (fig.  57, 
spectrum  8). 

(6)  The  band  is  much  better  seen  in  an  alcoholic  solution.  Prepare  the 
following  mixture  : — 150  c.c.  of  90-per-cent.  alcohol,  and  18  c.c.  of  50-per- 
cent, potash.     Take  about  5  c.c.  of  this  mixture  in  a  test-tube  and  boil  it. 


Fig.  58.— The  photographic  spectrum  of  haemoglobin  oxyhaemoglobin.    (Gamgee.) 

G  «K  LM       N    O 


Fig.  59.— The  photographic  spectrum  of  oxyhaemoglobin  and  methaemoglobin.    (Gamgee.) 

While  still  hot  drop  into  it  a  few  drops  of  undiluted  defibrinated  blood.  The 
fluid  then  shows  the  spectrum  of  alkaline  hsematin.  This  may  then  be  used 
for  the  next  experiment. 

6.  Haeinochromogen. — Add  ammonium  sulphide  to  the  solution  of  alkaline 
hgematin ;  the  colour  changes  to  red,  and  two  bands  are  seen,  one  between  D 


HiEMOGLOBIN  191 

andE,  and  the  other  nearly  coinciding  with  E  and  b  (fig.  57,  spectrum  9). 
The  spectrum  of  alkaline  haematin  reappears  for  a  short  time  after  vigorous 
shaking  witli  air. 

7.  Haematoporphyrin. — To  some  strong  sulphuric  acid  in  a  test-tube  add  a 
few  drops  of  undiluted  blood,  and  observe  the  spectrum  of  acid  haemato- 
porphyrin (iron-free  haematin)  (fig.  57,  spectrum  10).  Map  out  all  the  spectra 
you  see  on  a  chart. 

8.  The  photographic  Spectrum. — Haemoglobin  and  its  compounds  also 
show  absorption  bands  in  the  ultra-violet  portion  of  the  spectrum.  This 
portion  of  the  spectrum  is  not  visible  to  the  eye,  but  can  be  rendered  visible 
by  allowing  the  spectrum  to  fall  on  a  fluorescent  screen,  or  on  a  sensitive 
photographic  plate.  In  order  to  show  absorption  bands  in  this  part  of  the 
spectrum  very  dilute  solutions  of  the  pigment  must  be  used. 

In  order  to  demonstrate  these  bands,  the  telescope  of  a  large  spectroscope 
is  removed,  and  a  beam  of  sunlight  or  of  light  from  the  positive  pole  of  an 
arc  lamp  is  allowed  to  fall  on  the  slit  of  the  collimator.  The  spectrum  is 
focussed  on  a  fluorescent  screen.^  The  slit  is  then  opened  very  widely,  and 
the  coloured  solution  is  interposed  on  the  path  of  the  beam  falling  on  the 
slit. 

Oxyhaemoglobin  shows  a  band  (Soret's  band)  between  the  lines  G  and  H. 
In  haemoglobin,  carbonic  oxide  haemoglobin,  and  nitric  oxide  haemoglobin, 
this  band  is  rather  nearer  G.  Methaemoglobin  and  haematoporphyrin  show 
similar  bands. 

The  two  preceding  figures  show  the  '  photographic  spectra '  of  haemo- 
globin, oxyhaemoglobin,  and  methaemoglobin,  and  will  serve  as  examples  of 
the  results  obtained.  I  am  greatly  indebted  to  Prof.  Gamgee,  to  whom  we 
owe  most  of  our  knowledge  on  this  subject,  for  permission  to  reproduce  these 
two  specimens  of  his  numerous  photographs. 

0.  Preparation  of  Pure  Oxyhaemoglobin. — The  following  method  is  described 
in  Stirling's  '  Practical  Physiology.'  Centrifugalise  dog's  defibrinated  blood 
and  pour  off  the  serum.  Centrifugalise  again  with  physiological  saline 
solution  repeatedly  until  the  supernatant  fluid  contains  only  traces  of  protein. 
Mix  the  magma  of  corpuscles  with  two  or  three  volumes  of  water  saturated 
with  acid-free  ether ;  the  solution  becomes  clear.  Then  add  a  few  drops  of 
1-per-cent.  solution  of  acid  sodium  sulphate  till  the  mixture  looks  tinted  like 
fresh  blood,  owing  to  the  precipitation  of  the  stromata.  These  can  be 
separated  by  filtration.  Pour  off  the  clear  red  fluid :  cool  it  to  0°  C,  add 
one-fourth  of  its  volmne  of  absolute  alcohol  previously  cooled  to  0°  C.  Shake  • 
well,  and  then  let  the  mixture  stand  at  5°-15°  C.  for  24  hours.  As  a  rule 
the  whole  passes  into  a  glittering  crystalline  mass.  Filter  at  0°  C.  and  wash 
with  ice-cold  25-per-cent.  alcohol.  Eedissolve  the  crystals  in  a  small  quantity 
of  water,  and  recrystallise  as  before.  The  crystals  may  then  be  spread  on 
plates  of  porous  porcelain,  and  dried  in  a  vacuum  over  sulphuric  acid. 

'  Fluorescent  screens,  similar  to  those  in  common  use  in  observations  made 
with  Eontgen  rays,  may  be  made  by  coating  white  cardboard  with  barium  platino- 
cyanide. 


LESSON   XX 

SERUM 

1.  The  following  methods  of  precipitating  serum  globulin  should  be  per- 
formed : — 

(a)  Panum's  Method. — Dilute  serum  with  fifteen  times  its  bulk  of  water. 
It  becomes  cloudy  owing  to  partial  precipitation  of  the  serum  globulin.  Add 
a  few  drops  of  2-per-cent.  acetic  acid  ;  the  precipitate  becomes  more  abundant 
and  it  dissolves  in  excess  of  the  acid.     It  was  formerly  called  '  serum  casein.' 

(6)  Alexander  Schmidt's  Method. — Dilute  serum  with  twenty  times  its  bulk 
of  water  and  pass  a  stream  of  carbonic  acid  through  it.  A  fairly  abundant 
precipitate  of  serum  globulin  falls.  Let  it  settle  and  an  additional  precipitate 
can  be  obtained  from  the  decanted  liquid  by  treating  it  with  a  trace  of  acetic 
acid  (the  '  serum  casein '  mentioned  above).  Repeat  the  carbonic  acid 
method  without  dilution ;    no  precipitate  forms. 

(c)  By  Dialysis.  -  Put  some  serum  in  a  dialyser  with  distilled  water  in  the 
outer  vessel.  The  water  must  be  frequently  changed.  In  order  to  prevent 
decomposition  a  few  crystals  of  thymol  are  added.  In  a  day  or  two  the  salts 
have  passed  out ;  the  proteins  remain  behind  :  of  these  the  serum  albumin 
is  still  in  solution  ;  the  serum  globulin  is  in  part  precipitated,  as  it  requires 
a  small  quantity  of  salt  to  hold  it  in  solution.     (See  also  bottom  of  p.  193.) 

(d)  By  addition  of  Salts : — 

(i.)  Schmidt's  method.  Saturate  some  serum  with  sodium  chloride.  A 
precipitate  of  serum  globulin  is  produced. 

(ii.)  Hammarsten's  method.  Use  magnesium  sulphate  instead  of  sodium 
chloride.  A  more  abundant  precipitate  is  produced,  because  this  salt  is  a 
more  perfect  precipitant  of  serum  globulin  than  sodium  chloride.  In  order 
to  obtain  complete  saturation  with  these  salts  it  is  necessary  to  shake  the 
mixture  of  salt  and  serum  for  some  hours.^ 

(iii.)  Kauder's  method.  Half  saturate  serum  with  ammonium  sulphate. 
This  is  done  by  adding  to  the  serum  an  equal  volume  of  saturated  solution 
of  ammonium  sulphate.  This  precipitates  the  globulin.  Complete  satura- 
■  tion  with  the  salt  precipitates  the  albumin  also. 

2.  Heat  Coagulation. — Saturate  serum  with  magnesium  sulphate  and 
filter  off  the  precipitate  ;  preserve  the  filtrate  and  label  it  '  B:'  Wash  the 
precipitate  on  the  filter  with  saturated  solution  of  magnesium  sulphate  until 
the  washings  do  not  give  the  tests  for  albumin,'^  then  dissolve  the  precipitate 
by  adding  distilled  water.  It  readily  dissolves,  owing  to  the  salt  adherent 
to  it.     The  solution  is  opalescent.     Label  it  'A.' 

'  This  may  be  conveniently  done  by  a  shaking  machine  before  the  class  meets. 
2  On  account  of  the  prolonged  nature  of  these  operations,  they  must  necessarily 
be  performed  by  the  demonstrator  beforehand. 


SERUM  193 

Render  A  faintly  acid  with  a  drop  of  2-per-cent.  acetic  acid,  and  heat  in  a 
test-tube.  The  temperature  of  the  test-tube  may  be  raised  by  placing  it  in 
a  flask  of  water  gradually  heated  over  a  flame.  A  thermometer  is  placed  in 
the  test-tube,  and  should  be  kept  moving  so  as  to  ensure  that  all  parts  of  the 
^  liquid  are  at  the  same  temperature.  The  quantity  of  liquid  in  the  test-tube 
should  be  just  sufi&cient  to  cover  the  bulb  of  the  thermometer.  A  flocculent 
precipitate  of  coagulated  serum  globulin  separates  out  at  about  75°. 

Now  take  the  filtrate  B.  This  contains  the  serum  albumin.  Dilute  it 
with  an  equal  volume  of  water ;  render  it  faintly  acid  as  before,  testing  the 
reaction  with  litmus  paper.  Heat.  A  flocculent  precipitate  (a)  falls  at  about 
73°  C.  ;  filter  this  off;  note  that  the  filtrate  is  less  acid  than  that  from 
which  the  precipitate  has  separated,  or  it  may  even  be  alkaline.  If  so,  make 
it  faintly  acid  again^  and  heat;  a  precipitate  falls  at  77-79°  C.  (/3).  A  third 
precipitate  is  similarly  obtained  at  84-86°  C.  (y).  In  the  serum  of  the  ox, 
sheep,  and  horse  the  a  precipitate  is  absent  :  in  cold-blooded  animals,  the  (3 
and  y  varieties  are  absent. 

3.  Take  a  fresh  portion  of  B,  and  saturate  it  with  sodium  sulphate.  The 
serum  albumin  is  precipitated  (completely  after  a  long  shaking).  This  is 
due  to  the  formation  of  sodio-magnesium  sulphate.  B  was  already  saturated 
with  magnesium  sulphate  (MgSO,^  +  7H2O)  ;  on  adding  sodium  sulphate  a 
double  salt  (MgSO^.Na.^SO^  +  6H0O)  is  formed.  Shake  some  serum  with 
sodium  sulphate  alone.  A  small  precipitate  of  globulin  is  produced.'  Saturate 
another  portion  of  the  serum  with  sodio-magnesium  sulphate  ;  both  globulin 
and  albumin  are  precipitated. 

Of  the  methods  used  for  precipitating  serum  globulin  practically  only  two 
are  used  now.  There  are  Hammarsten's  and  Kauder's.  The  other  methods 
only  precipitate  the  globulin  incompletely.  Kauder's  method  is  rapid  and 
efficacious  :  if  the  globulin  is  filtered  off,  the  albumin  may  be  precipitated  in 
the  nitrate  by  complete  saturation  with  the  same  salt,  ammonium  sulphate. 
This  method  avoids  the  trouble  of  using  two  salts  as  described  under  3.  This 
last  method  is  instructive,  but  not  nearly  so  quick  as  Kauder's. 

With  regard  to  the  separation  of  serum  albumin  into  a,  jS,  and  y  varieties 
by  the  use  of  the  method  of  fractional  heat  coagulation,  it  must  be  men- 
tioned that  at  present  no  further  difference  has  been  shown  to  exist  between 
them,  and  the  opinion  has  been  very  freely  expressed  that  the  results 
obtained  are  not  trustworthy.  I  am  convinced  that  the  method  is  a  good  one, 
especially  as  in  other  cases  (see  Muscle)  the  proteins  so  separated  can  be 
shown  to  possess  other  differences.  In  the  case  of  serum,  however — and  the 
same  is  true  for  egg  albumin — the  matter  must  still  be  considered  subjudice. 

Recent  research  has  shown  that  sermn  globulin  is  not  a  single  protein. 
We  have  already  seen  that  the  precipitation  which  occurs  by  means  of 
dialysis  is  incomplete.  It  has  now  been  shown  that  serum  globulm  as 
'  salted  out '  by  means  of  the  sulphate  of  magnesium  or  ammonium  really 
consists  of  two  proteins  ;  one  of  these  (eu-globulin)  is  precipitable  by  dialysis : 
the  other  (pseudo-globulin)  is  not. 

*  That  is  at  room  temperature  ;  if  the  temperature  is  raised  to  36°  C.  sodium 
sulphate  acts  like  ammonium  sulphate  and  precipitates  all  the  proteins,  except 
peptone. 

0 


LESSON   XXT 

COAGULATION  OF  BLOOD 

1.  Effect  of  decalcifying  Agents  in  hindering  Coagulation. — From  an 
anaesthetised  dog  collect  samples  of  blood  from  the  carotid  artery,  into 
which  a  suitable  cannula  should  have  been  previously  inserted. 

(a)  Collect  the  first  sample  in  an  equal  volume  of  0*4  per  cent,  solution 
of  potassium  oxalate  made  with  physiological  salt  solution. 

(6)  Collect  the  second  sample  in  an  equal  volume  of  0-4  solution  of 
sodium  fluoride. 

(c)  Collect  the  third  sample  in  a  quarter  of  its  volume  of  10  per  cent, 
solution  of  sodium  citrate. 

In  all  three  cases  mix  thoroughly,  and  coagulation  is  hindered  owing  to 
decalcification,  as  explained  on  page  106. 

The  separation  of  the  plasma  from  the  corpuscles  may  be  most  readily 
carried  out  by  a  centrifugal  machine,  one  form  of  which  is  shown  in  the 
next  figure;  the  corpuscles  settle  and  the  supernatant  plasma  can  be 
then  pipetted  off.  Sedimentation  is  specially  rapid  in  the  case  of  citrate 
blood,  and  a  well-marked  layer  of  colourless  corpuscles  and  platelets  may 
usually  be  seen  on  the  top  of  the  mass  of  red  corpuscles. 

Oxalate  plasma  and  citrate  plasma  coagulate  on  the  restoration  of  the 
calcium  by  adding  a  few  drops  of  calcium  chloride  solution,  as  we  have 
already  seen  in  the  elementary  course  (p.  101).  Fluoride  plasma  does  not 
coagulate  unless  fibrin  ferment  (or  some  fluid  such  as  serum  which  contains 
fibrin  ferment  or  thrombin)  is  added  as  well  as  the  calcium  salt.  Fluoride 
plasma  thus  forms  a  convenient  test -fluid  for  fibrin  ferment. 

If  in  either  case  the  plasma  is  previously  heated  to  60°  C.  and  filtered, 
coagulation — that  is  to  say,  fibrin  formation — can  never  be  produced,  because 
its  mother-substance,  fibrinogen,  which  is  coagulated  by  heat  at  56°  C,  has 
been  destroyed  and  removed. 

2.  Influence  of  Leech  Extract  on  Coagulation. — The  same  dog  still  under  the 
anaesthetic  may  be  next  used  for  the  following  experiments  : — 

(a)  Draw  off  a  sample  of  blood  into  a  clean  test-tube,  and  note  the  time 
it  takes  to  clot. 

(6)  Draw  off  a  second  sample  into  about  half  its  volume  of  leech  extract, 
made  by  grinding  up  the  heads  of  about  twenty  leeches  in  20  c.c.  of  salt 
solution,  and  filtering.     This  remains  unclotted  for  hours  or  days. 

(c)  Inject  10  c.c.  of  the  extract  into  the  jugular  vein  of  the  animal,  and 
draw  off  samples  of  blood  from  time  to  time,  comparing  the  coagulation 
time  (which  gradually  lengthens)  with  that  of  specimen  a. 

{d)  Having  obtained  a  specimen  which  does  not  clot  at  all,  dilute  it  with 


COAGULATION  OF  BLOOD 


195 


salt  solution  and  pass  a  stream  of  carbon  dioxide  through  it.  Clotting  is  not 
produced  as  it  is  in  '  peptone  '  blood  (which  see).  In  order  to  produce  clot- 
ting, excess  of  serum,  or  some  fluid  containing  thrombin  must  be  added. 
The  action  of  leech  extract  is  mainly  due  to  the  fact  that  it  contains  anti- 
thrombin. 

(e)  The  experiments  described  under  d  may  be  repeated  with  leech 
extract  plasma,  obtained  from  the  blood  by  centrifugalising. 

(/)  Instead  of  leech  extract,  a  solution  of  its  active  principle  (hirudin) 
may  be  used.      This  produces  no  fall   of  blood  pressure,  and  so  contrasts 


Fiu.  tJO.— Centrifugal  machiue  as  made  by  Kumie.  Glass  vessels  containing  the  substances  t«j  be 
centrlfugalised  a:e  placed  within  the  six  metallic  tubes  whicli  hang  vertically  while  the  disc  is  at 
rest ;  when  tlie  machinery  is  set  going  they  fly  out  into  the  horizontal  position. 


with  what  occurs  in  '  peptone  '  injection.     Leech  extract  produces  a  very 
small  fall  of  arterial  pressure. 

3.  Influence  of  Commercial  Peptones  (Proteoses)  on  Coagulation.— For  the 
purpose  of  the  following  experiments  another  dog  must  be  employed. 

The  animal  having  been  anaesthetised  a  cannula  is  placed  in  the 
external  jugular  vein  for  the  injection  of  the  *  peptone.' 

The  carotid  artery  is  connected  to  a  mercurial  manometer  for  the 
registration  of  arterial  pressure. 

Another  convenient  artery  must  be  exposed  and  a  cannula  inserted  into 
it  for  the  collection  of  samples  of  blood. 

{a)  First  draw  off  a  sample  of  blood  and  note  its  coagulation  time. 

(5)  Draw  a  second  sample  into  a  strong  solution  of  commercial  peptone. 
The  coagulation  time  is  somewhat  longer  than  in  a. 

(c)  Then  inject  the  peptone  quickly,  so  that  the  animal  receives  0*3 
gramme  per  kilo,  of  body-weight.  Note  during  and  for  some  time  after  the 
injection  a  great  fall  in  arterial  blood  pressure.  This  has  been  shown  by 
the  oncometer  to  be  due  to  vascular  dilatation. 

o  2 


196  ESSENTIALS  OF  CHEMICAL    PHYSIOLOGY 

(d)  After  the  injection  draw  off  successive  samples,  and  note  the  great 
prolongation  of  the  coagulation  time  which  is  soon  produced. 

(e)  Dilute  some  of  the  blood  which  does  not  clot  with  twice  its  volume  of 
salt  solution,  and  pass  a  stream  of  carbonic  acid  through  the  mixture  ;  co- 
agulation soon  occurs. 

(/)  The  same  experiment  may  be  repeated  with  the  same  result,  if 
'  peptone  '  plasma  obtained  by  centrifugalising  is  used  instead  of  the  whole 
blood. 

{g)  Finally  bleed  the  animal  to  death,  collecting  the  blood  in  three 
successive  glass  cylinders.  Place  them  in  the  ice  chest,  and  examine  them 
a  few  days  or  a  week  later. 

The  first  lot  of  blood  collected  will  show  sedimentation  of  corpuscles, 
and  a  slight  clot  at  the  junction  of  the  corpuscles  and  supernatant  plasma — 
that  is,  at  the  place  where  the  white  corpuscles  and  platelets  lie. 

The  last  lot  of  blood  collected  shows  less  sedimentation,  and  will 
probably  have  clotted  throughout.  This  is  because  the  blood  removed  last 
has  been  diluted  by  tissue  lymph,  which  has  passed  into  the  blood -stream  in 
an  attempt  to  increase  the  volume  of  the  blood,  which  has  been  lessened 
by  the  previous  bleeding  ;  the  clot  produced  is  probably  due  to  the  action 
of  thrombokinase. 

The  middle  sample  will  show  something  intermediate  between  the  two 
extremes,  the  usual  state  of  things  being  clot  through  the  sediment,  and  the 
plasma  above  it  still  fluid.. 

4.  Intravascular  Coagulation. — A  solution  of  nucleo-protein  from  the 
thymus,  testis,  lymphatic  glands,  or  kidney  has  been  prepared  beforehand  by 
the  demonstrator.     It  may  be  prepared  in  one  or  two  ways. 

(a)  Wooldridge's  Method. — The  gland  is  cut  up  small  and  extracted  with 
water  for  twenty-four  hours.  Weak  acetic  acid  (0*5  c.c.  of  the  acetic  acid  of 
the  '  Pharmacopoeia  '  diluted  with  twice  its  volume  of  water  for  every  100  c.c. 
of  extract)  is  then  added  to  the  decanted  liquid.  After  some  hours  the  pre- 
cipitated nucleo-protem  (called  tissue-fibrinogen  by  Wooldridge)  falls  to  the 
bottom  of  the  vessel.  This  is  collected  and  dissolved  in  1 -per- cent,  sodium 
carbonate  solution. 

(6)  The  Sodiuin  Chloride  Method. — The  finely  divided  gland  is  ground 
up  in  a  mortar  with  about  an  equal  volume  of  sodium  chloride.  The  re- 
sulting viscous  mass  is  poured  into  excess  of  distilled  water.  The  nucleo- 
protein  rises  to  the  surface  of  the  water,  where  it  may  be  collected  and 
dissolved  as  before. 

A  rabbit  is  angesthetised,  and  a  cannula  inserted  into  the  external  jugular 
vein.  The  solutionis  injected  into  the  circulation  through  this.  The  animal 
soon  dies  from  cessation  of  respiration ;  the  eyeballs  protrude  and  the  pupils 
are  widely  dilated.  On  opening  the  animal  the  heart  will  be  found  still 
beating,  and  its  cavities  (especially  on  the  right  side)  distended  with  clotted 
blood.  The  vessels,  especially  the  veins,  also  are  full  of  clot.  The  blood  of 
the  portal  vein  is  usually  clotted  most.  If  a  dog  is  employed  instead  of  a  rabbit 
in  this  experiment,  coagulation  is  usually  confined  to  the  portal  area.  This 
is  related  to  the  greater  venosity  of  the  blood  in  this  situation.  If  venosity 
is  increased  in  any  other  area,  as  by  tetanising  the  muscles  of  one  leg,  clot- 
ting will  be  found  also  in  the  veins  of  this  region. 


LESSON   XXII 

MUSCLE   AND   NERVOUS   TISSUE 

1.  Hopkins's  Lactic  Acid  Test  (see  p.  185)  may  be  applied  as  follows.  Eemove 
one  hind  limb  of  a  pithed  frog.  Stimulate  the  sacral  plexus  of  the  other  side 
for  ten  minutes  with  a  strong  Faradic  current.  Then  amputate  the  other 
hind  limb.  Skin  both  legs,  and  chop  up  the  muscles  of  the  two  sides 
separately.  Pound  each  in  a  mortar  with  clean  sand  and  then  with  15  c.c. 
of  95-per-cent.  alcohol.  Transfer  the  mixture  to  a  beaker,  and  warm  in  the 
water-bath  for  a  few  minutes.  Filter,  and  evaporate  the  filtrate  to  dryness 
in  a  water-bath.  Extract  the  residue  with  about  5  c.c.  of  cold  water,  rubbing 
it  up  thoroughly  with  a  glass  rod.  Filter  and  boil  the  filtrate  in  a  test-tube 
for  about  a  minute  with  as  much  animal  charcoal  as  will  lie  on  a  threepenny- 
piece.  Filter  again  and  evaporate  the  filtrate  to  dryness  in  a  water-bath. 
Allow  the  residue  to  cool,  and  dissolve  it  by  shaking  in  5  c.c.  of  concentrated 
sulphuric  acid.  Transfer  this  to  a  dry  test-tube  ;  add  three  drops  of 
saturated  solution  of  copper  sulphate,  and  place  the  tube  in  boiling  water 
for  five  minutes.  Cool  and  add  2  drops  of  0*2-per-cent.  solution  of  thiophene 
in  alcohol ;  replace  the  tube  in  the  boiling  water.  A  cherry-red  colour 
develops  in  the  tube  containing  the  extract  from  tetanised  muscle,  but  not 
in  the  other. 

2  A  rabbit  has  been  killed  and  its  muscles  washed  free  from  blood  by  a 
stream  of  salt  solution  injected  through  the  aorta.  The  muscles  have  been 
quickly  removed,  chopped  up  small,  and  extracted  with  5-per-cent.  solution 
of  magnesium  sulphate.  This  extract  is  given  out.  It  will  probably  be 
faintly  acid.  The  acid  is  sarco-lactic  acid.  It  may  be  identified  by 
Ufifelmann's  (p.  185)  or  Hopkins's  reaction. 

3.  The  coagulation  of  musele  is  very  like  that  of  blood.  This  ma3'  be 
shown  with  the  salted  muscle  plasma  (the  extract  given  out)  as  follows  : 
Dilute  some  of  it  with  four  times  its  volume  of  water ;  divide  it  into  two 
parts ;  keep  one  at  40^  C.  and  the  other  at  the  ordinary  temperature.  Co- 
agulation— that  is,  formation  of  a  clot  of  myosin — occurs  in  both,  but  earliest 
in  that  at  40°  C. 

4.  Kemove  the  clot  of  myosin  from  3 ;  observe  it  is  soluble  in  10-per- 
cent, sodium  chloride,  and  also  in  0-2-per-cent.  hydrochloric  acid,  forming 
syntonin. 

5.  Make  an  extract  of  muscle  in  the  same  way,  using  a  small  quantity 
of  physiological  salt  solution  instead  of  the  strong  solution  of  magnesium 
sulphate  employed  in  the  foregoing  experiments.  Such  an  extract  contains 
the  two  principal  muscular  proteins,  viz.  paramyosinogen  (v.  Fiirth's  myosin) 


198  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

and  myosinogen  (v.  Fiirth's  myogen),  the  two  precursors  of  the  musele-clot 
or  myosin  (v.  Fiirth's  muscle-fibrin).  Small  quantities  of  other  proteins  also 
present  are  mainly  due  to  unavoidable  mixture  with  small  amounts  of  blood 
and  lymph. 

These  two  proteins  diflFer  in  temperature  of  heat  coagulation.  Take  the 
extract  and  heat  it  in  a  test-tube  within  a  water-bath  ;  at  4V  C.  para- 
myosinogen is  coagulated ;  filter  this  off  and  heat  the  filtrate  ;  at  56°  C. 
flocculi  of  the  coagulated  myosinogen  separate  out. 

6.  Paramyosinogen  is  precipitable  by  dialysis  and  is  a  true  globulin. 
Myosinogen  is  what  is  called  an  atypical  globulin,  and  corresponds  to  the 
pseudo-globulin  of  blood  serum  and  egg-white.  Though  readily  salted  out  of 
solution  like  paramyosinogen  it  is  not  precipitable  by  dialysis. 

7.  In  the  process  of  clotting,  such  as  occurs  in  rigor  mortis,  paramyosino- 
gen is  directly  converted  into  myosin ;  whereas  myosinogen  first  passes  into 
a  soluble  modification  (coagulable  by  heat  at  the  remarkably  low  temperature 
of  40°  C.)  before  myosin  is  formed.  This  is  shown  in  a  diagrammatic  way 
in  the  following  scheme  : — 

Proteins  of  the  living  muscle 


Paramyosinogen  Myosinogen 

"^\  Soluble  myosin 


Myosin 
(the  protein  of  the  muscle-clot). 

8.  When  a  muscle  is  gradually  heated,  at  a  certain  temperature  it  con- 
tracts permanently  and  loses  its  irritability.  This  phenomenon  is  known  as 
heat-rigor,  and  is  due  to  the  coagulation  of  the  proteins  in  the  muscle.  If  a 
tracing  is  taken  of  the  shortening,  it  is  found  that  the  first  shortening  occurs 
at  the  coagulation  temperature  of  paramyosinogen  (47°-50°  C),  and  if  the 
heating  is  continued  a  second  shortening  occurs  at  56°  C,  the  coagulation 
temperature  of  myosinogen.  If  frog's  muscles  are  used  there  are  three 
shortenings — namely,  at  40°,  47°,  and  56'  C;  frog's  muscle  thus  contains 
an  additional  protein  which  coagulates  at  40°  C.  This  additional  protein  is 
the  soluble  myosin  alluded  to  above,  some  of  which,  in  the  muscle  of  cold- 
blooded animals,  is  present  before  rigor  mortis  occurs. 

In  addition  to  the  proteins  mentioned,  there  is  a  small  quantity  of  nuclco- 
protein. 

9.  Involuntary  Muscle. — The  main  facts  just  described  for  voluntary  are 
true  also  for  involuntary  muscle.  The  chief  distinction  lies  in  the  quantity 
of  nucleo-protein,  which  is  more  abundant  in  those  forms  of  muscle  the 
fibres  of  which  are  least  different  from  the  mesoblastic  cells  from  which  all 
ultimately  originate.  This  may  be  readily  shown  by  the  following  simple 
experiment. 


MUSCLE   AND  NERVOUS   TISSUE 


199 


Take  equal  parts  of  voluntary  muscle,  heart  muscle,  and  plain  muscle 
(say  from  the  stomach  wall),  and  extract  each  for  the  same  time  with  equal 
amounts  of  0'15  per-cent.  solution  of  sodium  carbonate.  Filter  and  add  to 
each  filtrate   acetic  acid,  drop  by  drop.     The  extract   of  voluntary  muscle 


Vy 


D 


E 


F 


Fig.  61. — 1,  Absorption  spectrum  of  myohEematin,  as  seen  in  ninscle  rendered  transparent  by 
glycerin.    2,  Absorption  spectrnni  of  modified  myohaematin. 


gives  an  opalescence  only  ;  in  the  case  of  the  plain  muscle  there  is  an 
abundant  precipitate  ;  the  heart  muscle  gives  a  result  intermediate  between 
the  other  two. 

10.  Pigments  of  Muscle  : — 

{a)  Notice  the  difference  between  the  red  and  pale  muscles  of  the  rabbit. 


Fig.  62.— a  desiccator. 


(Gscheidlen.)    a,  glass  plate ;  b,  belljar  ;  c,  dish  of  sulphuric  add  ; 
d,  stand  for  capsules. 


(IS)  Examine  a  piece  of  red  muscle  {e.g.  the  diaphragm)  spectroscopically 
for  oxyhaemoglobin  (or  it  may  be  more  convenient  to  make  an  aqueous 
extract  of  the  muscle  and  examine  that). 

{c)  A  piece  of  the  pectoral  muscle  of  a  pigeon  has  been  soaked  in  glycerin. 


200 


ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 


Press  a  small  piece  between  two  glass  slides  and  place  it  in  fi'ont  of  the  spec- 
troscope. Observe  and  map  out  the  bands  of  myohsematin.  This  pigment  is 
doubtless  a  derivative  of  haemoglobin. 

(d)  Pieces  of  the  same  muscles  have  been  placed  in  ether  for  twenty-four 
hours.  The  ether  dissolves  out  a  yellow  lipochrome  from  the  adherent  fat. 
A  watery  fluid  below  contains  modified  myohaematin.  Filter  it ;  compare 
its  spectrum  with  that  of  hEemochromogen.  The  myohaematin  bands  are 
rather  nearer  the  violet  end  of  the  spectrum  (fig.  61,  spectrum  2)  than  those 
of  haemochromogen  (fig.  57,  spectrum  9). 

11.  Creatine  : — 

(a)  Take  some  of  the  red  fluid  described  in  10,  d,  and  let  it  evaporate  to 
dryness  in  a,  desiccator  over  sulphuric  acid  (fig.  62). 

In  a  day  or  two  crystals  of  creatine  tinged  with  myohaematin  separate  out. 

(b)  Take  an  aqueous  extract  of  muscle,  like  Ijiebig's  extract  or  beef-tea  ; 
add  baryta  water  to  precipitate  the  phosphates,  and  filter.  Eemove  excess 
of  baryta  by  a  stream  of  carbonic  acid ;  filter  off  the  barium  carbonate  and 
evaporate  the  filtrate  on  the  water-bath  to  a  thick  syrup.  Set  it  aside  to  cool, 
and  in  a  few  days  crystalline  deposits  of  creatine  will  be  found  at  the  bottom 
of  the  vessel.  These  are  washed  with  alcohol  and  dissolved  in  hot  water. 
On  concentrating  the  aqueous  solution  crystals  once  more  separate  out. 
which  may  be  still  further  purified  by  recrystallisation. 


NERVOUS   TISSUES 

The  chemical  investigation  of  nervous  tissues  is  not  well  adapted  to 
class  exercises  ;  still  it  may  not  be  uninteresting  to  state  briefly  the  princi- 
pal known  facts  in  relation  to  this  subject.  The  most  important  points 
which  any  table  of  analysis  will  show  are  :  (1)  the  large  percentage  of  water, 
especially  in  the  grey  matter ;  (2)  the  large  percentage  of  protein.  In  grey 
matter,  where  the  cells  are  prominent  structures,  this  is  most  marked,  and 
of  the  solids,  protein  material  here  comprises  more  than  half  of  the  total. 
The  following  are  some  analyses  which  give  the  mean  of  a  number  or 
observations  on  the  nervous  tissues  of  human  beings,  monkeys,  dogs,  and 
cats : — 


Percentage  of 



Water 

Solids 

Proteins  in 

SoUds 

Cerebral  grey  matter     . 

83-5 

16-5 

51 

„         white      „         .         .         . 

69-9 

30-1 

33 

Cerebellum 

79-8 

20-2 

42 

Spinal  cord  as  a  whole . 

71-6 

28-4 

31 

Cervical  cord          .... 

72-5 

27-5 

31 

Dorsal  cord 

69-8 

30-2 

28 

Lumbar  cord         .... 

72-6 

27-4 

33 

Sciatic  nerves        .... 

65-1 

34-9 

29 

The  most  important  protein  is  nude o -protein  ;  there  is  also  a  certain 
amount  of  glohvlin,  which,  like  the  paramyosinogen  of  muscle,  is  coagulated 
by  heat  at  the  low  temperature  of  47°  C.     A  certain  small  amount  of  neuro- 


MUSCLE  AND  NEKVOUS  TISSUE  201 

keratin  (especially  abundant  in  white  matter)  is  included  in  the  above  table 
with  the  proteins.  The  granules  in  nerve  cells  (Nissl's  bodies),  which  stain 
readily  with  methylene  blue,  are  nucleo-protein  in  nature.  The  next  most 
abundant  substances  are  of  a  fatty  nature ;  the  most  prominent  of  these  is 
the  phosphorised  fat  called  lecithin  (see  p.  25).  A  complex  substance 
called  protagon,  which  crystallises  out  on  cooling  a  hot  alcoholic  extract 
of  brain  or  other  nervous  structures  is  of  uncertain  composition.  Cerebrin 
is  a  term  which  probably  includes  several  substances,  which  are  nitro- 
genous glucosides ;  they  yield  on  hydrolysis  the  sugar  called  galactose 
(see  p.  18).  They  are  sometimes  called  cerebrosides.  There  are  other 
phosphorised  fats  as  well,  of  which  Tiejphalin  is  the  best  known.  The  crystal- 
line monatomic  alcohol  cliolestcr'm  (see  p.  93)  is  also  a  fairly  abundant  con- 
stituent of  nervous  structures,  especially  of  the  white  substance  of  Schwann. 
Finally  there  are  smaller  quantities  of  other  extractives,  and  a  small  pro- 
portion of  mineral  salts  (about  1  per  cent,  of  the  solids). 

Fresh  nervous  tissues  are  alkaline,  but  like  most  other  living  structures, 
they  turn  acid  after  death.  The  change  is  particularly  rapid  in  grey  matter. 
The  acidity  is  due  to  lactic  acid. 

Very  little  is  known  of  the  chemical  changes  nervous  tissues  under- 
go during  activity.  We  know  that  ox^^gen  is  very  essential,  especially 
for  the  activity  of  grey  matter ;  cerebral  anaemia  is  rapidly  followed  by  loss 
of  consciousness  and  death.  Waller  has  suggested  that  small  quantities  of 
carbonic  acid  are  produced  during  activity,  because  the  increase  in  the  action 
current  (detected  by  the  galvanometer)  which  occurs  after  a  nerve  has  been 
repeatedly  excited  is  very  like  the  increase  also  noted  on  the  application  of  small 
quantities  of  this  gas.  Waller's  suggestion  has  recently  been  confirmed  by 
direct  experiments  in  which  the  amount  of  carbon  dioxide  formed  has  been 
estimated.  Large  quantities  of  carbonic  acid  act  like  an  anaesthetic,  abolishing 
nervous  activity.  Of  all  parts  of  the  nervous  system,  the  cells  in  the  grey 
matter  are  those  which  most  readily  manifest  fatigue  ;  the  next  most  sensitive 
region  is  the  termination  of  nerves  in  such  endings  as  the  end-plates.  Fatigue 
in  a  meduUated  nerve-trunk  has  never  yet  been  experimentally  demonstrated ; 
Waller's  view  that  this  is  due  to  inter-nutritional  changes  between  the  axis 
cylinder  and  the  investing  medullary  sheath  can  hardly  be  considered 
proved,  for  it  is  just  as  difficult  to  demonstrate  fatigue  in  non-medullated 
nerves. 

Chemistry  of  Nerve-degeneration. — Mott  and  I  have  shown  that,  in  the 
disease  General  Paralysis  of  the  Insane,  the  marked  degeneration  that  occurs 
in  the  brain  is  accompanied  by  the  passing  of  the  products  of  degeneration 
into  the  cerebro-spinal  fluid.  Of  these,  nucleo-protein  and  choline— a  decom- 
position product  of  the  lecithin  (see  p.  26) — are  those  which  can  be  most  readily 
detected.  Choline  can  also  be  found  in  the  blood.  But  this  is  not  peculiar 
to  the  disease  just  mentioned,  for  in  various  other  degenerative  nervous 
diseases  (combined  sclerosis,  disseminated  sclerosis,  meningitis,  alcoholic 
neuritis,  beri-bei-i,  &c.)  choline  can  also  be  detected  in  these  situations.  The 
tests  employed  to  detect  choline  are  mainly  three :  (1)  The  fluid  is  diluted 
with  about  five  times  its  volume  of  alcohol  and  the  precipitated  proteins  are 
filtered  off.     The  filtrate  is  evaporated  to  dryness  at  40°  C.  and  the  residue 


202  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

dissolved  in  absolute  alcohol  and  filtered ;  the  filtrate  from  this  is  again 
evaporated  to  dryness,  and  again  dissolved  in  absolute  alcohol,  and  this 
should  be  again  repeated.  To  the  final  alcoholic  solution,  an  alcohohc  solution 
of  platinum  chloride  is  added,  and  the  precipitate  so  formed  is  allowed  to 
settle  and  is  washed  with  absolute  alcohol  by  decantation ;  the  precipitate  is 
then  dissolved  in  15-per-cent.  alcohol,  filtered,  and  the  filtrate  is  allowed  to 
slowly  evaporate  in  a  watch-glass  at  40°  C.  The  crystals  can  then  be  seen 
with  the  microscope.  They  are  recognised  not  only  by  their  yellow  colour 
and  octahedral  form,  and  by  their  solubility  in  water  and  15-per  cent,  alcohol, 
but  also  by  the  fact  that  on  incineration  they  yield  31  per-cent.  of  platinum 
and  give  off  the  odour  of  trimethylamine.  There  is  a  danger  of  mistaking 
such  cr^fstals  for  those  obtained  from  the  chlorides  of  potassium  and  am- 
monium ;  but  the  presence  of  such  contaminations  may  be  minimised  by  the 
use  of  alcohol  as  water-free  as  possible.  (2)  The  following  test,  however, 
is  entirely  distinctive  of  choline  and  leads  to  no  risk  of  confusion  with  other 
substances.  The  final  alcoholic  solution  prepared  as  above  is  evaporated  to 
dryness,  and  the  residue  taken  up  with  water,  to  this  is  added  a  strong 
solution  of  iodine  (2  grammes  of  iodine  and  G  grammes  of  potassium 
iodide  in  100  c.c.  of  water).  In  a  few  minutes  dark-brown  prisms  of 
choline  periodide  are  formed.  These  look  very  like  haemin  crystals.  If 
the  slide  is  allowed  to  stand  so  that  the  liquid  gradually  evaporates,  the 
crystals  slowly  disappear,  and  their  place  is  taken  by  brown  oily  droplets^ 
but  if  a  fresh  drop  of  the  iodine  solution  is  added  the  crystals  slowly  form 
once  more.  (3)  A  physiological  test,  namely  the  lowering  of  arterial  blood- 
pressure  (partly  cardiac  in  origin,  and  partly  due  to  dilatation  of  peripheral 
vessels)  which  a  saline  solution  of  the  residue  of  the  alcoholic  extract  pro- 
duces :  this  fall  is  abolished,  or  even  replaced  by  a  rise  of  arterial  pressure,  if 
the  animal  has  been  atropinised.  Such  tests  have  already  been  shown  to  be 
of  diagnostic  value  in  the  distinction  between  organic  and  so-called  functional 
diseases  of  the  nervous  system. 

A  similar  condition  can  be  produced  artificiall}^  in  animals  by  a  division 
of  large  nerve-trunks  ;  and  is  most  marked  in  those  animals  in  which  the 
degenerative  process  is  at  its  height  as  tested  histologically  by  the  Marchi 
reaction.^  A  chemical  analysis  of  the  nerves  themselves  was  also  made.  A 
series  of  cats  was  taken,  both  sciatic  nerves  divided,  and  the  animals  subse- 
quently killed  at  intervals  varying  from  1  to  106  days.  The  nerves  remain 
practically  normal  as  long  as  they  remain  irritable  :  that  is,  up  to  about  three 
days  after  the  operation.  They  then  show  a  progressive  increase  in  the  per- 
centage  of  water,  and  a  progressive  decrease  in  the  percentage  of  phosphorus 
until  degeneration  is  complete.  When  regeneration  occurs,  the  nerves  return 
approximately  to  their  previous  chemical  condition.  ■  One  chemical  feature 
of  degeneration  is  the  replacement  of  phosphorised  by  non-phosphorised  fat. 
When  the  Marchi  reaction  disappears  in  the  later  stages  of  degeneration,  the 


'  The  Marchi  reaction  is  the  black  staining  that  the  medullary  sheath  of 
degenerated  nerve  fibres  shows  when,  after  being  hardened  in  Miiller's  fluid,  they  are 
treated  with  Marchi's  reagent,  a  mixture  of  Miiller's  fluid  and  osmic  acid.  Healthy 
nerve  fibres  are  but  little  affected  by  the  reagent,  but  degenerated  myelin  is 
blackened  like  the  fat  of  normal  adipose  tissue. 


MUSCLE   AND   NERVOUS  TISSUE 


203 


non-phosphoriscd  fat  has  been  absorbed.  This  absorption  occurs  earUer  in 
tlie  peripheral  nerves  than  in  the  central  nervous  system.  The  non-phos- 
phorised  fat  of  degenerated  myelin  is  also  either  richer  in  olein,  or  the  olein 
is  more  loosely  combined  than  in  the  healthy  medullary  sheath;  hence 
the  deeper  reaction  with  osmic  acid  even  in  the  presence  of  chromic  acid 
as  in  the  Marchi  test.  The  following  table  gives  details  of  these  experi- 
ments : — 


Cat's  sciatic  nerves 

Condition 
of  blood 

( Minimal 

i  traces  of  cho- 

{ line  present 

j  Choline  more 
( abundant 

f  Choline  abun- 
1  dant 

j  Choline  much 
jless 

( Choline  al- 
J  most  disap- 
( peared 

Condition  of 
nerves 

■ 
—                     Water 

Normal                                 65-1   ^ 
1  to  3  days  after  section    64-5 

4  to  G                „                   69-3 

8                        „                   68-2 
10                        „                   70-7 
13                        „                   71-3 

25-27                 „                  72-1 
29                        „                   72-5 

44                        „                   72-6 
100  to  106            „                   G6-2 

1 

Solids 

34-9 
35-5 

30-7 

31-8 
29-3 

28-7 

27-9 
27-5 

27-4 
33-8 

Percentage  of 

phosphoras  in 

solids 

1-1 
0-9 

0-9 

0-5 
0-3 

U-2 

traces 
0 

0 
0-9 

( Nerves  irritable 

J  and  histologically 

i  healthy 
Irritability  lost : 
degeneration  be- 

,  ginning 
Degeneration  well 

■  shown  by  Marchi 
( reaction 

Marchi  reaction 
still  seen,  but  ab- 

■  sorption  of  de- 
generated fat  has 
set  in 
Absorption  of  fat 

■  practically  com- 
(plete 

j  Keturn  of  function, 
t  nerves  regenerated 

The  foregoing  figures  relate  to  the  peripheral  portions  of  the  nerves.  Noll 
has  also  shown  that  the  phosphorised  fat  diminishes  somewhat  in  the  central 
ends  of  cut  nerves  due  to  '  disuse  atroph3^' 

Further,  it  has  been  found  that  in  human  spinal  cords  in  which  a  unilateral 
degeneration  of  the  pyramidal  tract  has  been  produced  by  a  lesion  in  the 
opposite  hemisphere,  and  which  gives  the  Marchi  reaction,  there  is  a  similar 
increase  of  water  and  diminution  of  phosphorus  on  the  degenerated  side. 

Cerebro-spinal  Fluid. — This  plays  the  part  of  the  lymph  of  the  central 
nervous  system,  but  differs  considerably  from  all  other  forms  of  lymph.  It 
is  a  very  watery  fluid,  containing,  besides  some  inorganic  salts  similar  to 
those  of  the  blood,  a  trace  of  protein  matter  (globulin)  and  a  small  amount 
of  a  reducing  substance,  the  nature  of  which  was  for  a  long  time  uncertain 
but  which  seems  now  to  have  been  proved  to  be  sugar.  It  contains  the 
merest  trace  of  choline ;  but  this  is  not  devoid  of  significance,  for  this  fact 
taken  in  conjunction  with  another — namely,  that  physiological  saline  solution 
will  extract  from  perfectly  fresh  nervous  matter  a  small  quantity  of  choline — 
shows  us  that  lecithin  is  not  a  stable  substance,  but  is  constantly  breaking 
down  and  building  itself  up  afresh;  in  fact,  undergoing  the  process  called 
metabolism.  This  is  most  marked  in  the  most  active  region  of  the  brain — 
vi7..  the  grey  matter. 


LESSON   XXIII 

UREA   AND   CHLORIDES  IN  URINE 

ESTIMATION   OF    UREA 

If  albumin  is  present  it  must  be  first  separated  by  boiling  after  acidulation 
with  acetic  acid  if  necessary,  and  filtering  off  the  flakes  of  coagulated  protein. 
The  hypobromite  method  of  estimation  (see  p.  141)  holds  its  own,  as  it  is  easy 
and  sufficiently  exact  for  clinical  purposes.  It  has  entirely  replaced  the  older 
method  of  Liebig  (titration  with  mercuric  nitrate),  which  is  now  of  purely 
historical  interest. 

When  absolute  accuracy  is  necessary,  one  or  other  of  many  recently 
introduced  methods  must  be  employed.  We  shall  be  content  with  describing 
two  of  these. 

(a)  Folin's  Method. — This  depends  on  the  fact  that  urea  is  decomposed 
quantitatively  into  ammonia  and  carbonic  acid  by  boiling  with  magnesium 
chloride  solution.  The  ammonia  is  estimated  by  distillation  into  standard 
acid  and  subsequent  titration. 

Analysis. — Three  c.c.  of  urine,  20  grammes  of  magnesium  chloride  and2c.c. 
of  concentrated  hydrochloric  acid  are  boiled  in  a  flask,  closed  by  a  cork  through 
which  a  glass  tube  20  centimetres  in  height  passes.  This  acts  as  a  reflux 
condenser.  The  boiling  is  continued  for  25  to  30  minutes.  After  diluting 
with  water  the  mixture  is  then  transferred  to  a  litre  flask,  7  c.c.  of  20-per- 
cent, caustic  soda  are  added,  and  the  ammonia  is  distilled  off  and  estimated 
as  described  in  the  final  operation  in  Kjeldahl's  method  (see  Appendix,  p.  235). 
Every  c.c.  of  decinormal  ammonia  in  the  distillate  corresponds  to  3  milli- 
grammes of  urea.  A  small  correction  has  to  be  made  for  the  ammonia 
present  as  such  in  the  original  urine. 

(h)  Method  of  Mbrner  and  Sjbqvist. — The  following  reagents  are  necessary: 

i.  A  saturated  solution  of  barium  chloride  containing  5  per  cent,  of 
barium  hydrate. 

ii.  A  mixture  of  ether  and  alcohol  in  proportion  1 :  2. 

iii.  The  apparatus,  &c.,  necessary  for  carrying  out  Kjeldahl's  method  of 
estimating  nitrogen  (see  p.  235). 

Analysis. — Five  c.c,  of  urine  are  mixed  with  5  c.c.  of  the  barium  mixture 
and  100  c.c.  of  the  mixture  of  ether  and  alcohol.  By  this  means  all  nitrogenous 
substances  except  urea  are  precipitated.  Twenty-four  hours  later  this  is  filtered 
off,  and  the  precipitate  is  washed  with  50  c.c.  of  the  ether-alcohol  mixture, 
the  filter-pump  being  used  to  accelerate  the  process.  The  washings  are 
added  to  the  filtrate ;  a  little  magnesia  is  added  to  this  to  drive  off  ammonia. 


UKEA  AND   CHLORIDES   IN   URINE  205 

The  alcohol  and  ether  are  then  driven  off  at  a  temperature  of  55°  C;  and 
evaporation  is  continued  at  this  temperature  until  the  volume  of  the  residue 
is  10-15  c.c.  The  nitrogen  in  this  is  estimated  by  Kjeldahl's  method.  The 
nitrogen  found  is  multiplied  by  2*143,  and  the  result  is  the  amount  of  urea. 

PKEPARATION  OF  UREA  FROM  URINE 

(1)  Evaporate  the  urine  to  a  small  bulk.  Add  strong  pure  nitric  acid  in 
excess,  keeping  the  mixture  cool  during  the  addition  of  the  acid.  Pour  off 
the  excess  of  fluid  from  the  crystals  of  urea  nitrate  which  are  formed ;  strain 
through  muslin  and  press  between  filter  paper.  Add  to  the  dry  product 
barium  carbonate  in  large  excess.  This  forms  barium  nitrate  and  sets  the 
urea  free.  Mix  thoroughly  with  sufficient  methylated  spirit  to  form  a  paste. 
Dry  on  a  water-bath  and  extract  with  alcohol ;  filter  ;  evaporate  the  filtrate 
on  a  water-bath  and  set  aside.  The  urea  crystallises  out,  and  may  be  de- 
colourised by  animal  charcoal  and  purified  by  recrystallisation. 

(2)  The  following  method  is  well  adapted  for  the  preparation  of  micro- 
scopic specimens  of  urea  and  urea  nitrate  :  Take  20  c.c.  of  lu-ine  ;  add 
baryta  mixture  (see  footnote  3,  p.  5)  until  no  further  precipitate  is  produced  ; 
filter,  evaporate  the  filtrate  to  a  thick  syrup  on  the  water-bath,  and  extract 
with  alcohol ;  pour  off  and  filter  the  alcoholic  extract ;  evaporate  it  to  dry- 
ness on  the  water-bath  and  take  up  the  residue  with  water.  Place  a  drop 
of  the  aqueous  solution  on  a  slide  and  allow  it  to  crystallise ;  crystals  of 
urea  separate  out.  Place  another  drop  on  another  slide  and  add  a  drop  of 
nitric  acid  ;  crystals  of  urea  nitrate  separate  out. 

ESTIMATION    OF    CHLORIDES 

The  chlorides  in  the  urine  consist  of  those  of  sodium  and  potassium,  the 
latter  only  in  small  quantities.  The  method  adopted  for  the  determination 
of  the  total  chlorides  consists  in  their  precipitation  by  a  standard  solution 
of  silver  nitrate  (Mohr's  method). 

The  following  solutions  must  be  prepared  : — 

Standard  silver  nitrate  solution.  Dissolve  29*075  grammes  of  fused 
nitrate  of  silver  in  a  litre  (1,000  c.c.)  of  distilled  water  :  1  c.c.  =-0'01  gramme 
of  sodium  chloride. 

(a)  Saturated  solution  of  neutral  potassium  chromate. 

Analysis. — Take  10  c.c.  of  urine ;  dilute  with  100  c.c.  of  distilled  water. 

Add  to  this  a  few  drops  of  the  potassium  chromate  solution. 

Drop  into  this  mixture  from  a  burette  the  standard  silver  nitrate  solu- 
tion ;  the  chlorine  combines  with  the  silver  to  form  silver  chloride,  a  white 
precipitate.  AVhen  all  the  chlorides  are  so  precipitated,  silver  chromate  (red 
in  colour)  goes  down,  but  not  while  any  chloride  remains  in  solution.  The 
silver  nitrate  must  therefore  be  added  until  the  precipitate  has  a  pink  tinge. 

From  the  amount  of  standard  solution  used,  the  quantity  of  sodium 
chloride  in  10  c.c.  of  urine,  and  thence  the  percentage,  may  be  calculated. 

Sources  of  Error  and  Correctio7is. — A  high-coloured  urine  may  give  rise 
to  difficulty  in  seeing  the  pink  tinge  of  the  silver  chromate  :  this  is  overcome 
by  diluting  the  urine  more  than  stated  in  the  preceding  paragraph. 

One  c.c.  should  always  be  subtracted  from  the  total  number  of  c.c.  of  the 


206  ESSENTIALS   OF  CHEMICAL   PHYSIOLOaY 

silver  nitrate  solution  used,  as  the  urine  contains  small  quantities  of  certain 
compounds  more  easily  precipitable  than  the  chromate. 

To  obviate  such  sources  of  error  the  following  modification  of  the  test, 
as  described  by  Sutton,  may  be  used  :  10  c.c.  of  urine  are  measured  into  a 
thin  porcelain  capsule  and  1  gramme  of  pure  ammonium  nitrate  added  ;  the 
whole  is  then  evaporated  to  dryness,  and  gradually  heated  over  a  small 
spirit  lamp  to  low  redness  till  all  vapours  are  dissipated  and  the  residue 
becomes  white.  It  is  then  dissolved  in  a  small  quantity  of  water,  and  the 
carbonates  produced  by  the  combustion  of  the  organic  matter  neutralised  by 
dilute  acetic  acid ;  a  few  grains  of  pure  calcium  carbonate  to  remove  all  free 
acid  are  then  added,  and  one  or  two  drops  of  potassium  chromate.  The 
mixture  is  then  titrated  with  decinornial  silver  solution  (16*966  gr.  of  silver 
nitrate  per  litre)  until  the  end  reaction,  a  pink  colour,  appears.  Each  c.c.  of 
silver  solution  represents  0'005837  gr.  of  salt ;  consequently,  if  12*5  c.c.  have 
been  used,  the  weight  of  salt  in  the  10  c.c.  of  urine  is  0-07296  gr.,  or  0'7296 
per  cent.  If  5*9  c.c.  of  urine  are  taken  for  titration,  the  number  of  c.c.  of 
silver  solution  used  will  represent  the  number  of  parts  of  salt  per  1,000  parts 
of  urine. 


LESSON   XXIV 

ESTIMATION  OF  PHOSPHATES  AND   SULPHATES  IN   URINE 

ESTIMATION    OF    PHOSPHATES 

The  phosphoric  acid  in  the  urine  is  combined  with  soda,  potash,  hine,  and 
magnesia. 

(a)  Estimation  of  the  total  pliosphates. 

For  this  purpose  the  following  reagents  are  necessary  : — 

i.  A  standard  solution  of  uranium  nitrate.  The  uranium  nitrate  solution 
contains  85*5  grammes  in  a  litre  of  water  ;  1  c.c.  corresponds  to  0*005  gramme 
of  phosphoric  acid  (P^O^). 

ii.  Acid  solution  of  sodium  acetate.  Dissolve  100  grammes  of  sodium 
acetate  in  900  c.c.  of  water ;  add  to  this  100  c.c.  of  glacial  acetic  acid. 

iii.  Solution  of  potassium  ferrocyanide. 

Method. — Take  50  c.c.  of  urine.  Add  5  c.c,  of  the  acid  solution  of  sodium 
acetate.^     Heat  the  mixture  to  80°  C. 

Run  into  it  while  hot  the  standard  uranium  nitrate  solution  from  a 
burette  until  a  drop  of  the  mixture  gives  a  distinct  brown  colour  with  a  drop 
of  potassium  ferrocyanide  placed  on  a  porcelain  slab.  Read  off  the  quantity 
of  solution  used  and  calculate  therefrom  the  percentage  amount  of  phosphoric 
acid  in  the  urine. 

Another  indicator  which  may  be  used  is  cochineal  tincture,  a  few  drops  of 
which  may  be  added  to  the  mixture.  A  change  of  colour  from  red  to  green 
is  the  sign  of  the  end  of  the  reaction. 

(6)  Estimation  of  the  'phosphor ic  acid  combined  with  lime  and  magnesia 
(alkaline  earths). 

Take  200  c.c.  of  urine.  Render  it  alkaline  with  ammonia.  Lay  the  mixtm-e 
aside  for  twelve  hours.  Collect  the  precipitated  earthy  phosphates  on  a  filter ; 
wash  with  dilute  ammonia  (1  in  3).  Wash  the  precipitate  off  the  filter  with 
water  acidified  by  a  few  drops  of  acetic  acid.  Dissolve  with  the  aid  of  heat, 
adding  a  little  more  acetic  acid  if  necessary.  Add  5  c.c.  of  the  acid  solution 
of  sodium  acetate.  Bring  the  volume  up  to  50  c.c,  and  estimate  the  phos- 
phates in  this  volumetrically  by  the  standard  uranium  nitrate  as  before. 
Subtract  the  phosphoric  acid  combined  with  the  alkaline  earths  thus  obtained 
from  the  total  quantity  of  phosphoric  acid,  and  the  difference  is  the  amount 
of  acid  combined  with  the  alkalis,  soda  and  potash. 

'  In  using  uranium  nitrate  it  is  imperative  that  sodium  acetate  should 
accompany  the  titration  in  order  to  avoid  the  possible  occurrence  of  free  nitric  acid 
in  the  solution.     If  uranium  acetate  is  used,  it  may  ha  omitted. 


208  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

(c)  Instead  of  uranmm  nitrate,  a  standard  solution  of  uranium  acetate 
may  be  used.  The  directions  for  the  making  of  these  standard  solutions  will 
be  found  in  Sutton's  *  Volumetric  Analysis.'  As  a  rule,  it  is  less  troublesome, 
and  not  much  more  expensive,  to  purchase  standard  solutions  ready  made. 


ESTIMATION    OF    SULPHATES 

The  sulphates  in  the  urine  are  of  two  kinds  :  the  pre-formed  sulphates — 
viz.  those  of  soda  and  potash — and  the  combined  or  ethereal  sulphates. 

(a)  For  the  determination  of  the  total  amount  of  sulphuric  acid  (SO^)  (i.e. 
pre-formed  and  combined  sulphuric  acid  together)  in  the  urine,  one  of  two 
methods  is  adopted : — 

1.  Volumetric  method. 

2.  Gravimetric  method. 

Both  methods  will  be  given  here :  the  former  is,  however,  better  suited 
for  class  experiments. 

1.  Volu7netric  Determination. — This  process  consists  in  adding  to  a 
given  volume  of  the  urine  a  standard  solution  of  chloride  of  barium  so  long 
as  a  precipitate  of  barium  sulphate  is  formed. 

The  following  solutions  are  necessary  : — 

i.  Standard  barium  chloride  solution :  30*5  grammes  of  crystallised 
chloride  of  barium  in  a  litre  of  distilled  water ;  1  c.c.  of  this  solution  corre- 
sponds to  O'Ol  gramme  of  sulphuric  acid  (SOg). 

ii.  Solution  of  sulphate  of  potash  :  20  per  cent. 

iii.  Pure  hydrochloric  acid. 

Method. — 100  c.c.  of  urine  are  taken  in  a  flask.  This  is  rendered  acid  by 
5  c.c.  of  hydrochloric  acid,  and  boiled.  The  combined  sulphates  are  thus 
converted  into  ordinary  sulphates,  and  give  a  precipitate  like  them  with 
barium  chloride.  The  chloride  of  barium  solution  is  allowed  to  drop  into 
this  mixture  as  long  as  any  precipitate  occurs,  the  mixture  being  heated  before 
every  addition  of  barium  chloride  to  it.  After  adding  5  to  8  c.c.  of  the 
standard  solution,  allow  the  precipitate  to  settle  ;  pipette  off  a  few  drops  of 
the  clear  supernatant  fluid  into  a  watch-glass ;  add  to  it  a  few  drops  of  the 
standard  barium  chloride  solution.  If  any  precipitate  occurs,  return  the 
whole  to  the  flask  and  add  more  barium  chloride  ;  again  allow  the  precipitate 
to  settle,  and  test  as  before  ;  go  on  in  this  waj^  until  no  more  barium  sulphate 
is  formed  on  the  addition  of  barium  chloride. 

Excess  of  barium  chloride  must  also  be  avoided ;  when  only  a  trace  of 
excess  is  present  a  drop  of  the  clear  fluid  removed  from  the  precipitate  gives 
a  cloudiness  with  a  drop  of  the  potassium  sulphate  solution  placed  on  a 
glass  plate  over  a  black  ground.  If  more  than  a  cloudiness  appears,  too 
large  a  quantity  of  bariinn  chloride  has  been  added  and  the  operation  must 
be  repeated.  From  the  quantity  of  barium  chloride  solution  used,  the  per- 
centage of  sulphuric  acid  in  the  urine  is  calculated. 

2.  Oravimetric  Determination  {i.e.  by  weight). — This  method  consists  in 
weighing  the  precipitate  of  barium  sulphate  obtained  by  adding  barium 
chloride  to  a  known  volume  of  urine  ;  100  parts  of  sulphate  of  barium 
correspond  to  34-33  parts  of  sulphuric  acid  (SO3). 


ESTIMATION   OF  PHOSPHATES  AND   SULPHATES   IN  URINE     209 

Method  (Salkowski). — 100  c.c.  of  urine  are  taken  in  a  beaker.  This  is 
boiled  with  5  c.c.  of  hydrochloric  acid  as  before. 

Chloride  of  barium  is  added  till  no  more  precipitate  occurs. 

The  precipitate  is  collected  on  a  small  filter  of  known  ash,  and  washed 
with  hot  distilled  water  till  no  more  barium  chloride  occurs  in  the  filtrate — 
i.e.  until  the  filtrate  remains  clear  after  the  addition  of  a  few  drops  of  sulphuric 
acid.  Then  wash  with  hot  alcohol,  and  afterwards  with  ether.  Remove 
the  filter,  and  place  it  with  its  contents  in  a  platinum  crucible.  Heat  to 
redness.  Cool  over  sulphuric  acid  in  a  desiccator ;  weigh,  and  deduct  the 
weight  of  the  crucible  and  filter  ash  ;  the  remainder  is  the  weight  of  barium 
sulphate  formed. 

Error. — When  the  experiment  is  carried  out  as  above  there  is  a  slight 
error  from  the  formation  of  a  small  quantity  of  sulphide  of  barium.  This 
may  be  corrected  as  follows: — After  the  platinum  crucible  has  become  cool 
add  a  few  drops  of  pure  sulphuric  acid  (H2SO4).  The  sulphide  is  converted 
into  sulphate.     Heat  again  to  redness  to  drive  off  excess  of  sulphuric  acid. 

(b)  The  following  is  Salkowski's  method  of  estimating  the  coinhined 
suljyliuric  acid — that  is,  the  amount  of  SO.,  in  ethereal  sulphates  : — 100  c.c. 
of  urine  are  mixed  with  100  c.c.  of  alkaline  barium  chloride  solution,  which  is 
a  mixture  of  two  volumes  of  solution  of  barium  hydrate  with  one  of  barium 
chloride,  both  saturated  in  the  cold.  The  mixture  is  stirred,  and  after  a  few 
minutes  filtered  :  100  c.c.  of  the  filtrate  ( =  50  c.c.  of  urine)  are  acidified  with 
10  c.c.  of  hydrochloric  acid,  boiled,  kept  at  100°  C.  on  the  water-bath  for  an 
hour,  and  then  allowed  to  stand  till  the  precipitate  has  completely  settled ; 
if  possible,  it  should  be  left  in  this  way  for  twenty-four  hours.  The  further 
treatment  of  this  precipitate  ( =  combined  sulphates)  is  then  carried  out  as  in 
the  last  case. 

Calculation. — 233  parts  of  barium   sulphate    correspond  to  98  parts  of 

HoSO^,  or  80  parts  of  SO3  or  32  parts  of  S.    To  calculate  the  H.^SO^,  multiply 

98 
the  weight  of  barium  sulphate  by  --^  =  0*421 ;  to  calculate  the  SO3,  multiply 

Zoo 
on  Qo 

by  ry^=  0-343;    to  calculate  the  S  multiply  bvJ;"  =0-137.       This    method 
233  "  233 

of  calculation  applies  to  the  gravimetric  estimation  both  of  total  sulphates 
and  of  combined  sulphates. 

(c)  To  obtain  the  amount  of  pre-formed  sulphuric  acid,  subtract  the 
amount  of  combined  SO.,  from  the  total  amount  of  S0.(.  The  difference  is 
the  pre-formed  SO3. 

Example :  100  c.c.  of  urine  gave  0-5  gramme  of  total  barium  sulphate. 

80 
This  multiplied  by  ^^"  =  0-171  gr.  =  total  SO.,.     Another  100  c.c.  of  the  same 
2oo 


urine  gave  0*05  gr.  of  barium  sulphate  from  ethereal  sulphates  ;  this  multi- 
plied by  ^^  =  0-017   gr.   of    combined    SO3. 
=-.0-171-0-017  =  0-154  gr.  of  pre-formed  SO,. 


plied  by  ^^  =  0-017   gr.   of    combined    SO3.      Total    SO, -combined     SO, 
2oo 


LESSON   XXV 
URIC  ACID  AND   CREATININE 

1.  Preparation  of  Pure  Uric  Acid. — If  one  wishes  to  prepare  pure  uric  acid, 
the  solid  urine  of  a  reptile  or  bird,  which  consists  principally  of  the  acid 
amnionmni  salt,  should  be  selected ;  one  has  not  then  to  separate  any  pig- 
ment. It  is  boiled  with  10-per-cent.  caustic  soda  or  ammonia,  diluted,  and 
then  allowed  to  stand.  The  clear  fluid  is  decanted  and  poured  into  a  large 
excess  of  water  to  which  10  per  cent,  of  hydrochloric  acid  has  been  added ; 
after  twenty-four  hours  crystals  of  uric  acid  are  deposited.  These  may  be 
purified  by  washing,  re-solution  in  soda,  and  re-precipitation  by  acid. 

2.  Estimation  of  Uric  Acid  (Hopkins's  method). — The  following  reagents 
are  required  :  Pure  chloride  of  ammonium,  finely  powdered. 

A  wash-bottle  containing  a  filtered  saturated  solution  of  the  same  salt. 

A  twentieth  normal  solution  of  potassium  permanganate  made  by  dis- 
solving 1-581  grammes  of  permanganate  in  a  litre  of  water. 

Measure  100  c.c.  of  urine  into  a  beaker  of  about  150  c.c.  capacity. 

Add  to  this  25  grammes  (approximately  weighed)  of  ammonium  chloride, 
stirring  briskly  till   all    the   salt   is   dissolved.     Now   add  2  c.c.  of  strong 
ammonia,  and  allow  the  mixture  to  stand  until  the  precipitate  of  ammonium 
urate,  which  rapidly  forms,  has  wholly  settled  to  the  bottom  of  the  beaker 
its  subsidence  is  promoted  by  occasional  brisk  stirring. 

Adjust  a  small  filter  paper  (7  cm.  diam.)  in  a  funnel  of  such  size  that  only 
a  small  margin  of  glass  projects  above  the  edge  of  the  folded  paper,  and 
transfer  to  this  the  ammonium  urate  precipitate. 

Filtration  should  not  be  commenced  until  the  precipitate  has  settled 
satisfactorily.  The  precipitate  should  be  as  far  as  possible  retained  in  the 
beaker  until  the  greater  part  of  the  clear  liquid  has  filtered  through  ;  finally 
transfer  the  whole  to  the  filter  with  the  help  of  a  wash -bottle  containing 
saturated  ammonium  chloride  solution.  After  the  filter  has  thoroughly 
drained,  wash  the  precipitate  twice  again  with  the  same  solution. 

While  the  last  washings  are  running  through  the  paper,  distiUed  water 
should  be  heated  to  boiling  in  a  wash-bottle  provided  with  a  fine  jet.  The 
funnel  containing  the  filter  is  now  held  horizontally  over  a  small  porcelain 
basin  (of  about  50  c.c.  capacity)  and  the  precipitate  washed  into  the  latter 
with  a  jet  of  hot  water,  the  filter  itself  being  afterwards  opened  out  over  the 
basin  in  order  that  any  urate  adhering  to  its  folds  may  be  washed  off.  Not 
more  than  20-30  c.c.  of  water  need  be  emploj^ed  in  this  transference  :  if  much 
more  has  been  used  the  liquid  should  be  concentrated  over  the  water-bath  at 


URIC  ACID  AND  CREATININE  211 

this  stage.  A  little  strong  HCl  (1  c.c.)  is  next  added  to  the  contents  of  the 
basin,  and  the  whole  is  then  heated  over  a  burner  until  it  just  reaches  the 
boiling  point.     It  is  then  set  aside  for  the  uric  acid  to  crj'stallise  out. 

If  the  mixture  is  artificially  cooled  all  the  uric  acid  will  separate  out  in 
two  hours,  otherwise  it  is  best  allowed  to  stand  overnight  or  longer. 

The  crystals  are  filtered  off  through  a  very  small  filter  paper  (4  cm. 
diam.) ;  the  filtrate  is  received  into  a  graduated  cylinder  so  that  the 
amount  of  mother  liquid  may  be  noted  (see  below).  The  uric  acid  is  next 
washed  with  cold  distilled  water  until  free  from  chlorides.  It  is  unnecessary 
to  transfer  the  whole  to  the  filter  ;  the  greater  part  may  be  washed  by  decanta- 
tion.  Such  of  the  crystals  as  are  upon  the  filter  are  now  washed  back  into 
the  basin  (best  by  the  aid  of  hot  water)  and  the  whole  quantity  is  dissolved 
by  heating  to  boiling  with  1  c.c.  of  10-per-cent.  sodium  carbonate  solution 
and  as  much  distilled  water  as  the  basin  will  safely  hold. 

The  solution  is  transferred  to  a  {-litre  Erlenmeyer  flask,  which  should  be 
marked  roughly  at  100  c.c.  The  solution  is  made  up  to  this  mark  with  dis- 
tilled water,  and  cooled  to  the  temperature  of  the  room. 

Twenty  c.c.  of  strong  sulphuric  acid  are  added  to  the  contents  of  the 
flask,  and  the  mixture  shaken  and  titrated  with  the  standard  permanganate 
solution. 

During  the  addition  of  the  standard  solution  the  liquid  in  the  flask  should 
be  kept  in  vigorous  movement.  It  will  be  found  that  at  first  the  disappear- 
ance of  the  pink  colour  is  so  rapid  that  each  drop  as  it  is  added  is  decolorised 
before  it  has  time  to  diffuse  through  the  whole  liquid.  The  first  instan- 
taneous appearance  of  a  diffused  flush  throughout  the  solution  indicates  the 
end  point  of  the  reaction.  This  colour  rapidly  disappears,  but  it  will  be  found 
that  the  effect  of  adding  further  quantities  of  permanganate  after  the  end 
point  has  been  passed  is  quite  different  from  the  effect  before  the  end  point 
was  reached ;  each  drop  is  now  able  to  diffuse  throughout  the  fluid. 

For  each  c.c.  of  the  solution  necessary  to  produce  the  end  point  just 
described  0'00375  gramme  of  uric  acid  is  present.  To  the  value  so  obtained 
1  mgm.  must  be  added  for  each  15  c.c.  of  the  mother  liquor  from  which  the 
crystals  separated.  Thus  the  uric  acid  from  100  c.c.  of  a  sample  of  urine 
used  up  18-5  c.c.  of  the 'standard  permanganate  solution.  The  mother  liquor 
filtered  from  crystals  measured  25  c.c. 

18-5  X  -00375  =  -0694  gr. 

•001  X  ^^       =  -0017 
15 

Total       =  -OTli 

The  urine  contained  71  mgms.  uric  acid  per  100  c.c. 

3.  Estimation  of  Creatinine. — The  following  colorimetric  method  (Folin's) 
is  now  generally  employed  for  the  estimation  of  creatinine ;  and  with  a 
slight  modification  it  may  also  be  used  for  the  estimation  of  creatine.  It  is 
based  on  the  red  colour  which  Jaffe  showed  develops  when  an  alkaline  solu- 
tion of  picric  acid  is  added  to  a  solution  of  creatinine  ;  this  is  coiiipared 
wdth  the  colour  of  a  standard  solution  of  potassium  bichromate,  the  tint  of 
the  two  fluids  being  almost  identical.     If  creatine  has  to  be  estimated,  this  is 

p3 


212  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

first  transformed  into  creatinine  by  boiling  with  hydrochloric  acid.  Tlie 
apparatus  and  reagents  necessary-  are  : — 

1.  A  colorimeter  consisting  of  two  tubes,  the  height  of  the  column  in 
which  can  be  read  by  graduations  in  tenths  of  a  millimetre. 

2.  A  half  normal  solution  of  potassium  bichromate   (24*5  grammes  per 
Htre). 

3.  A  saturated  solution  of  picric  acid. 

4.  10  per  cent,  caustic  soda. 

To  perform  the  analysis  one  tube  of  the  colorimeter  is  filled  with  the 
bichromate  solution  up  to  the  height  of  8  mm.  Ten  c.c.  of  urine  are  measured 
into  a  half-litre  flask,  15  c.c.  of  the  picric  acid  solution  and  5  c.c.  of  the 
caustic  soda  solution  added,  and  then  water  until  the  total  volume  of  the 
mixture  is  500  c.c.  This  solution  is  poured  into  the  second  tube  of  the  colori- 
meter to  such  a  height  (which  is  read  off)  that,  on  looking  down  through  it 
the  intensity  of  the  colour  is  the  same  as  that  in  the  standard  tube  by  its 
side.  The  amount  necessary  for  this  corresponds  exactly  to  8'1  milligi-ammes 
of  creatinine.  From  this  the  total  in  the  sample  of  urine  can  be  easily 
calculated. 


LESSON   XXVI 

THE   PIGMENTS   OF  THE    URINE 

The  urinary  pigments  are  numerous,  and  have  from  tnne  to  time  been 
described  under  different  names  by  various  observers. 

1.  Urochrome. — This  is  the  essential  yellow  pigment  of  the  urine.  The 
word  was  originally  introduced  by  Thudichum,  and  the  substance  he  obtained 
is  now  recognised  to  have  been  a  mixture  of  several  pigments,  of  v/hich, 
however,  the  essential  yellow  pigment  formed  a  large  proportion.  Garrod's 
method  of  separating  it  from  the  urine  is  as  follows : — 

The  urine  is  saturated  with  ammonium  sulphate  and  filtered.  The 
filtrate  contains  the  pigment ;  this  is  shaken  with  alcohol.  The  alcohol 
separates  readily  from  the  saline  mixture,  and  as  it  does  so  dissolves  out 
much  of  the  urochrome.  By  repeated  extraction  all  the  pigment  passes  into 
solution  in  the  alcohol.  The  alcoholic  solution  is  diluted  with  water,  and 
the  mixture  again  saturated  with  ammonium  sulphate.  The  alcohol  con- 
taining the  pigment  in  solution  again  separates  out.  The  second  alcoholic 
solution  is  made  faintly  alkaline  with  ammonia  and  evaporated  to  dryness. 
The  residue  is  extracted  once  or  twice  with  acetic  ether,  and  then  again 
dissolved  in  strong  alcohol.  Finally  the  alcohol  is  concentrated  till  it  is 
deep  orange  in  tint,  and  poured  into  an  equal  volume  of  ether.  The  pure 
pigment  is  by  this  means  precipitated  as  an  amorphous  brown  powder. 

Urochrome  shows  no  absorption  bands.  As  already  stated  (p.  143),  it  is 
probably  an  oxidation  product  of  urobilin. 

2.  Urobilin.— Urobilin  is  a  derivative  of  the  blood-pigment,  and  is  identical 
with  stercobilin  (see  pp.  92,  143).  Probably  both  reduction  aud  hydration 
occur  in  its  formation.  It  is  very  like  the  substance  named  hydrobilirubin 
by  Maly,  which  he  obtained  by  the  action  of  sodium  amalgam  on  bilirubin. 
The  following  formulae  show  the  relationship  between  these  allied  pig- 
ments : — 

Haematin   .         .  ....     Cg^Hg^N^O^Fe 

Bilirubin C32H3,N,0,' 

Hydrobilirubin C-jaH^oNiO^ 

Urobilin  is  probably  a  further  stage  in  reduction. 

Normal  urine  contains  but  little  urobilin  ;  what  is  present  is  chiefly  in 
the  form  of  a  colourless  chromogen,  which  by  oxidation  is  converted  into 
urobilin.     In  numerous  pathological  conditions  urobilin  is  abundant. 

The  following  are  the  two  methods  introduced  by  Garrod  and  Hopkins 
for  its  separation  from  the  urine  : — 

(a)  The  urine  is  first  saturated  with  ammonium  chloride,  and  the  urate 


214  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

so  precipitated  is  filtered  off.  The  filtrate  is  then  acidified  with  sulphuric 
acid  and  saturated  with  ammonium  sulphate.  This  causes  a  precipitate  of 
urobilin,  which  maj-  be  collected  and  dissolved  in  water.  The  aqueous 
solution  is  again  saturated  with  ammonium  sulphate,  and  the  pigment 
is  thus  precipitated  in  a  state  of  purity. 

(b)  The  urates  are  first  removed,  then  the  urine  is  acidified  and  saturated 
with  ammonium  sulphate  as  before.  The  urobilin  is  then  extracted  from  the 
mixture  by  shaking  it  with  a  mixture  of  chloroform  and  ether  (1  : 2)  in  a 
lai'ge  separating  funnel.  The  ether-chloroform  extract  is  then  rendered 
faintly  alkaline  and  shaken  with  distilled  water,  and  the  urobilin  passes  into 
solution  in  the  water.  The  aqueous  solution  is  now  once  more  saturated 
with  ammonium  sulphate  and  slightly  acidified ;  it  then  once  more  yields  its 
pigment  to  ether-chloroform. 

By  means  of  either  of  these  methods  urobilin  is  obtained  in  a  pure  con- 
dition ;  even  normal  urine  will  give  some,  for  the  chromogen  is  partly  con- 
verted into  the  pigment  by  the  acid  employed. 

Urobilin  dissolved  in  alcohol  exhibits  a  green  fluorescence,  which  is 
greatly  increased  by  the  addition  of  zinc  chloride  and  ammonia.  It  shows  a 
well-marked  absorption  band  between  b  and  F,  slightly  overlapping  the  latter 
(fig.  63,  spectrum  4). 

Urobilin,  like  most  animal  pigments,  shows  acidic  tendencies  and  forms 
compounds  with  bases  ;  it  is  liberated  from  such  combinations  by  the 
addition  of  an  acid. 

If  urobilin  is  dissolved  in  caustic  potash  or  soda,  and  sufficient  sulphuric 
or  hydrochloric  acid  is  added  to  render  the  liquid  faintly  acid,  a  turbidity  is 
produced.  This  turbid  liquid  shows  an  additional  band  in  the  region  of  the 
E  line  (fig.  63,  spectrum  6),  which  is  probably  due  to  the  special  light  absorp- 
tion exercised  by  fine  particles  of  urobilin  in  suspension.  It  whoUy  dis- 
appears when  the  precipitate  is  filtered  off,  and  when  it  is  re-dissolved  the 
ordinary  band  alone  is  visible. 

3.  Uroerythrin. — This  is  the  colouring  matter  of  j)ink  urate  sediments. 
It  may  be  separated  from  the  sediment  as  follows  : — The  deposit  is  washed 
with  ice-cold  water,  dried,  and  placed  in  absolute  alcohol.  The  alcohol, 
though  a  solvent  for  uroerythrin,  does  not  extract  it  from  the  urates.  The 
alcohol  is  poured  off,  and  the  deposit  dissolved  in  warm  water.  From  this 
solution  the  pigment  is  easily  extracted  by  amy  lie  alcohol. 

Uroerythrin  has  a  great  affinity  for  urates,  with  which  it  appears  to  form 
a  loose  compound.  Its  solutions  are  rapidly  decolorised  by  light.  Bpectro- 
scopically  it  shows  two  rather  ill -defined  bands  (fig,  63,  spectrmii  7).  It 
gives  a  green  colour  with  caustic  potash,  and  red  or  pink  with  mineral  acids. 
Uroerythrin  appears  to  be  a  small  but  constant  constituent  of  urine.  Its 
origin  and  relationship  to  other  pigments  are  unknown. 

4.  Haematoporphyrin. — This  also  occurs  in  small  quantities  in  normal 
urine.  In  some  pathological  conditions,  especially  after  the  administration 
of  certain  drugs  (e.g.  sulphonal),  its  amount  is  increased.  Its  amount  is 
stated  to  increase  when  the  urine  stands  ;  this  points  to  the  existence  of  a 
colourless  chromogen.  It  may  be  separated  from  the  urine  as  follows  : — 
Caustic    alkali    is    added    to   the    urine ;  this    causes  a  precipitate  of  phos- 


THE  PIGMENTS  OF  THE   URINE 


215 


phates,  which  carries  down  the  pigment  with  it :  the  pigment  may  be  dis- 
solved out  with  chloroform.  The  chloroform  is  evaporated,  the  residue 
washed  with  alcohol,  and  finally  dissolved  in  acidified  alcohol.  Urines  rich 
in  the  pigment  yield  it  easily  to  acetic  ether  or  to  amylic  alcohol. 


i 


cEg 


I 


Fig.  63.— Chart  of  absorption  spectra  :  1,  acid  liaematoporphyriii ;  2,  alkaline  haBUiatoporphyrin  ; 
3,  haematoporpbyrin  as  found  sometimes  in  urate  sediments  ;  4,  acid  urobilin,  concentrated  ; 
5,  acid  urobilin," dilute  ;  6,  the  E  band  spectrum  of  urobilin  ;  7,  uroerythrin  ;  8,  urorosein  con- 
centrated—on dilution  the  band  shrinks  rapidly  from  redward  end.    (After  F.  G.  Hopkins.) 

When  the  urine  is  sufficiently  rich  in  the  pigment,  the  bands  shown  are 
those  of  alkaline  htematoporphyrin  (fig.  63,  spectrum  2).  On  adding  sulphuric 
acid  the  spectrum  of  acid  hyematoporphyrin  is  seen  (fig.  63,  spectrum  1). 
Occasionally  urate  sediments  are  pigmented  with  a  form  of  the  pigment  which 
shows  a  two-banded  spectrum,  very  like  that  of  oxyhgemoglobin   (fig.  63, 


216  ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 

spectrum  3)  ;  by  treatment  with  dilute  mineral  acids  this  changes  immedi- 
ately to  the  spectrum  of  acid  haematoporphyrin. 

5.  Chromogens  in  Urine. — In  addition  to  the  chromogens  of  urobilin  and 
haematoporphyrin  alluded  to  in  the  foregoing  paragraphs  there  are  others,  of 
which  the  following  may  be  mentioned  : — (a)  Indoxyl.—The  origin  of  this 
substance  from  indole  is  mentioned  on  p.  154.  It  is  easily  oxidised  to 
indigo-blue  or  indigo-red. 

2C,H,<^2^>CH  f  0,  =  C,3H,  <^,^>C:C<^^>C,H,  +  2H,0. 

[indoxyl]  [indigo-blue] 

Indigo-red    is    isomeric    with    indigo-blue,  its    structural   formula    being 

CO  C  OTT 

C(,H^<^-g->C:C<p*TT    >N.      It  is  very  rare  for  the  urine  to  be  actually 

pigmented  with  indigo,  for  the  urinary  indoxyl  is  excreted  as  a  conjugated 
sulphate  which  resists  oxidation.  When  the  urine  is  mixed  with  an  equal 
volume  of  hydrochloric  acid,  indoxyl  is  liberated  from  the  sulphate.  A 
solution  of  a  hypochlorite  is  then  added  drop  by  drop,  when  indigo-blue  is 
formed,  and  on  shaking  the  mixture  with  chloroform  the  indigo-blue  passes 
into  the  chloroform.  (Jaffe.)  This  test,  however,  is  not  a  very  good  one,  for 
the  hypochlorite  solution  has  always  to  be  freshly  prepared,  and  even  then  a 
small  excess  will  cause  the  colour  to  disappear  owing  to'  oxidation  of  the 
indigo,  and  it  is  difficult  to  hit  off  the  exact  amount  to  give  the  reaction.  A 
better  test  for  indoxyl-sulphuric  acid  (indican)  consists  in  adding  10  c.c.  of 
a  20-per-cent.  solution  of  lead  acetate  to  50  c.c.  of  urine,  and  filtering  the 
mixture.  The  filtrate  is  shaken  with  an  equal  volume  of  hydrochloric  acid 
(containing  0*2  to  0*4  per  cent,  of  ferric  chloride)  and  a  few  c.c.  of  chloroform. 
The  indigo-blue  passes  into  the  chloroform  (Obermayer.)  (6)  Shatoxyl. — 
When  skatoxyl  is  given  by  the  mouth  it  passes  into  the  urine,  and  yields 
skatoxyl-red  on  oxidation,  (c)  Urorosein  is  distinct  from  indigo -red.  It  is 
produced  from  its  chromogen  by  the  action  of  mineral  acids.  It  frequently 
appears  when  urine  is  treated  with  strong  hydrochloric  acid  and  allowed  to 
stand,  but  it  appears  more  readily  when  an  oxidising  agent  is  added  as  well. 
It  is  readily  soluble  in  amylic  alcohol,  but  not  in  ether.  The  chromogen  is 
precipitated  by  saturation  with  ammonium  sulphate.  The  colour  is  de- 
stroyed by  alkalis.  It  shows  an  absorption  band  between  the  D  and  E  lines 
(fig.  63,  spectrum  8). 

6.  Pathological  Pigments. — The  most  frequently  appearing  of  abnormal 
pigments  are  those  of  blood  and  bile.  The  urine  may  contain  accidental 
pigments  due  to  the  use  of  drugs  (rhubarb,  senna,  logwood,  santonin)  ;  in 
carbolic  acid  poisoning  pyrocatechin  and  hydrochinon  are  chiefly  responsible 
for  the  greenish-brown  colour  of  the  urine,  which  increases  on  exposure  to 
the  air.  The  black  or  dark-brown  pigment  called  melanin  may  pass  into 
the  urine  in  cases  of  melanotic  sarcoma.     For  alcaptonuria  see  p.  169. 


APPENDIX 


HEMACYTOMETERS 

Gowers's  Haemacytometer. — The  enumeration  of  the  blood  corpuscles  is 
readily  effected  bj^  the  haemacytometer  of  Gowers.  This  instrument  consists 
of  a  glass  slide  (fig.  64,  C),  the  centre  of  which  is  ruled  into  ^  millimetre 
squares  and  surrounded  by  a  glass  rim  i  millimetre  thick.  It  is  provided 
with  measuring  pipettes  (A  and  B),  a  vessel  (D)  for  mixing  the  blood  with  a 
saline  solution  (sulphate  of  soda  of  specific  gravity  1015),  a  glass  stirrer  (E), 
and  a  guarded  needle  (F). 


Fig.  64.— Ha?macvtometer  of  Sir  W.  Gowers. 


Nine  hundred  and  ninety-five  cubic  millimetres  of  the  saline  solution  are 
measured  out  by  means  of  A,  and  then  placed  in  the  mixing  jar;  5  cubic 
millimetres  of  blood  are  then  drawn  from  a  puncture  in  the  finger  by  means 
of  the  pipette  B,  and  blown  into  the  solution.  The  two  fluids  are  well 
mixed  by  the  stirrer,  and  a  small   drop  of  this  diluted  mixture  placed  in  the 


218 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


centre  of  the  slide  C,  a  cover  glass  is  gently  laid  on  (so  as  to  touch  the  drop, 
which  thus  forms  a  layer  4  millimetre  thick  between  the  slide  and  cover 
glass),  and  pressed  down  by  two  brass  springs.  In  a  few  minutes  the 
corpuscles  have  sunk  to  the  bottom  of  the  layer  of  fluid,  and  rest  on  the 
squares.  The  number  on  ten  squares  is  then  counted,  and  this  multiplied 
by  10,000  gives  the  number  in  a  cubic  millimetre  of  blood.  The  average 
number  of  red  corpuscles  in  each  square  ought  therefore  in  normal  human 
blood  to  be  45-50. 

Differential  counts  to  show  the  relative  proportions  of  the  varieties  of 
leucocytes  are  made  in  appropriately  stained  specimens. 

Oliver's  Haeniacytoiiieter.  — The  following  method,  devised  by  Dr.  George 


FiO.  G5.— Uliver'.s  liiumacytouieter. 


APPENDIX 


219 


Oliver,  is  a  ready  way  of  determining  the  total  number  of  corpuscles.  It  is, 
however,  not  possible  to  determine  the  relative  proportion  of  red  and  white 
corpuscles  by  this  means. 

The  linger  is  pricked,  and  the  blood  allowed  to  flow  into  the  small 
capillary  pipette  (fig.  65,  a)  until  it  is  full.  This  is  washed  out  by  the 
dropping  tube  b  into  a  graduated  flattened  test-tube,  c,  with  Haycm's  fluid.' 
The  graduations  of  the  tube  are  so  adjusted  that  with  normal  blood  con- 
taining 5,000,000  coloured  corpuscles  per  cubic  millimetre,  the  light  of  a 
small  wax  candle  placed  at  a  distance  of  9  feet  from  the  eye  in  a  dark  room 
is  just  transmitted  as  a  fine  bright  line  when  looked  at  through  the  tube  held 
edgeways  between  the  fingers  (d)  and  filled  up  to  the  100  mark  of  the  gradua- 
tion. If  the  number  of  corpuscles  is  less  than  normal,  less  of  the  diluting 
solution  is  required  for  the  light  to  be  transmitted  ;  if  above  the  normal,  more 
of  the  Hayem's  fluid  must  be  added.  The  tube  is  graduated,  so  as  to  indicate 
in  percentages  the  decrease  or  increase  of  corpuscles  per  cubic  millimetre  as 
compared  with  the  normal  standard  of  100  per  cent. 

H^MOGLOBINOMETERS 

Gowers's  Haemoglobinoineter. — ^The  apparatus  consists  of  two  glass  tubes, 
C  and  D,  of  the  same  size.  D  contains  glycerin  jelly  tinted  with  carmine  to 
a  standard  colour — viz.,  that  of  normal  blood  diluted  100  times  with  distilled 
water.     The  finger  is  pricked  and  20  cubic  millimetres  of  blood  are  measured 


Fig.  66.— HaRmoglohinometer  of  Sir  W.  Gowers. 


out  by  the  capillary  pipette,  B.  This  is  blown  out  into  the  tube  C,  and  diluted 
with  distilled  water,  added  drop  by  drop  from  the  pipette  stopper  of  the 
bottle.  A,  until  the  tint  of  the  diluted  blood  reaches  the  standard  colour.  The 
tube  C  is  graduated  into  100  parts.     If  the  tint  of  the  diluted  blood  is  the 


'  Sodium  sulphate  5  grammes,  sodium    chloride    1 
0-5  grm.,  distilled  water  200  c.c. 


':rm.,    mercuric    chloride 


220 


ESSENTIALS   OF  CHEMICAL  PHYSlOLOaY 


same  as  the  standard  when  the  tube  is  filled  up  to  the  graduation  100,  the 
quantity  of  oxyhsemoglobin  in  the  blood  is  normal.  If  it  has  to  be  diluted 
more  largely,  the  oxyhsemoglobin  is  in  excess ;  if  to  a  smaller  extent,  it  is 
less  than  normal.  If  the  blood  has,  for  instance,  to  be  diluted  up  to  the 
graduation  50,  the  amount  of  haemoglobin  is  only  half  what  it  ought  to  be — 
50  per  cent,  of  the  normal — and  so  for  other  percentages. 

Haldane's  Haemoglobinometer  is  more  frequently  used.  Instead  of  tinted 
gelatin,  the  standard  of  comparison  is  a  sealed  tube  filled  with  a  solution  of 
carbonic  oxide  haemoglobin  of  known  strength.  This  keeps  unchanged  for 
years.  A  stream  of  coal  gas  is  passed  through  the  blood  to  be  examined. 
This  converts  all  the  haemoglobin  present  into  carboxyhaemoglobin  ;  this  is 
then  diluted  with  water  to  match  the  standard. 

Von  Fleischl's  Haemometer. — The  apparatus  (fig.  67)  consists  of  a  stand 
bearing  a  white  reflecting  surface  (S)  and  a  platform.     Under  the  platform  is 


Fig.  67. — Von  Fleischl's  liiemomete 


a  slot  carrying  a  glass  wedge  stained  red  (K)  and  moved  by  a  wheel  (R). 
On  the  platform  is  a  small  cylindrical  vessel  divided  vertically  into  two  com- 
partments, a  and  a' . 

Fill  with  a  pipette  the  compartment  a'  over  the  wedge  with  distilled 
water.     Fill  about  a  quarter  of  the  other  compartment  {a)  with  water. 

Prick  the  finger  and  fill  the  short  capillary  pipette  provided  with  the 
instrument  with  blood.  Dissolve  this  in  the  water  in  compartment  a,  and 
fill  it  up  with  distilled  water.  Having  arranged  the  reflector  (S)  to  throw 
artificial  light  vertically  through  both  compartments,  look  down  through 
them,  and  move  the  wedge  of  glass  by  the  milled  head  (T)  until  the  colour 
in  the  two  is  identical.  Read  off  the  scale,  which  is  so  constructed  as  to  give 
the  percentage  of  hyemoglobin. 

Oliver's  Haemoglobinometer. — This  method  consists  in  compaiing  a  speci- 


APPENDIX 


221 


men  of  blood,  suitably   diluted    with   water  in  a    shallow   white   palette, 
with  a  number  of  standard  tests   very  carefully   prepared   by    the  use  of 


riG.68.-01iver;sh^moglobiuometer:  a,  standard  gradations ;  &,  lancet ;  <•  capillary 
measuring  pipette  ;  d,  mixing  pipette  ;  e,  blood  cell  and  cover  glass. 

Lovibond's  coloured  glasses.     The   capillary  pipette  c  (fig.  68)  is  first  filled 
with  Dlood  obtained  by  .pricking  the  finger.     This   is  washed   with  water 


222  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

by  the  mixing  pipette  d  into  the  blood  cell  e ;  the  cell  is  then  just  filled  with 
water,  and  the  blood  and  water  thoroughly  mixed  by  the  handle  of  c  being 
used  as  a  stirrer.  The  cover  glass  is  then  adjusted,  when  a  small  bubble 
should  form,  a  clear  sign  that  the  cell  has  not  been  overfilled.  The  cell  is 
then  placed  by  the  side  of  the  standard  gradations,  and  the  eye  quickly 
recognises  its  approximate  position  on  the  scale.  The  camera  tube  provided 
with  the  instrument  will  more  accurately  define  it.  Artificial  light  should 
be  used. 

If  it  is  proved  that  the  blood  solution  is  matched  in  depth  of  colour  by 
one  of  the  standard  grades,  the  observation  is  at  an  end  ;  but  if  the  tint  is 
higher  than  one  grade,  but  lower  than  another,  the  blood  cell  is  placed 
opposite  to  the  former,  and  riders  (not  shown  in  the  illustration)  are  added 
to  complete  the  observation.  The  standard  gr&dations  are  marked  in  per- 
centages, 100  per  cent,  being  taken  as  normal. 

*  "Worth '  of  the  Corpuscles. — If  the  percentage  of  haemoglobin  is  100,  and  the 

100 
percentage   number   of  corpuscles  is  100  also,  then  the  quotient      ^  =  1  is 

100 

taken  as  the  normal.  This  varies  in  health  from  0*95  to  1'05  in  men,  and 
from  09  to  1  in  women.  This  quotient  has  been  termed  the  '  worth  '  of  the 
corpuscle. 

Specific  Gravity  of  Blood.— Of  the  numerous  methods  introduced  for  taking 
the  specific  gravity  of  fresh  blood,  that  of  Hammerschlag  is  the  simplest.  A 
drop  of  blood  from  the  finger  is  placed  in  a  mixture  of  chloroform  and 
benzene.  If  the  drop  falls,  add  chloroform  till  it  just  begins  to  rise  ;  if  the  drop 
rises,  add  benzene  till  it  just  begins  to  fall.  The  fluid  will  then  be  of  the 
same  specific  gravity  as  the  blood.  Take  the  specific  gravity  of  the  mixture 
in  the  usual  waj'  with  an  accurate  hydrometer. 

Schmalz's  capillary  pycnometer  is  more  accurate. 


POLARISATION    OF    LIGHT 

If  an  object,  such  as  a  black  dot  on  a  piece  of  white  paper,  be  looked  at 
through  a  crystal  of  Iceland  spar,  two  black  dots  will  be  seen ;  and  if  the 
crystal  be  rotated,  one  black  dot  will  move  round  the  other,  which  remains 
stationary.  That  is,  each  ray  of  light  entering  such  a  crystal  is  split  into  two 
rays,  which  travel  through  the  crystal  with  different  velocities,  and  conse- 
quently one  is  more  refracted  than  the  other.  One  ray  travels  just  as  it 
would  through  glass  ;  this  is  the  ordinary  ray,  the  ray  which  gives  the 
stationary  image  ;  the  other  ray  gives  the  movable  image  when  the  crystal 
is  rotated  ;  the  ordinary  laws  of  refraction  do  not  apply  to  it,  and  it  is  called 
the  extraordinary  ray.  Both  rays  are  of  equal  brilliancj'.  In  one  direction, 
however,  that  of  the  optic  axis  of  the  crystal,  a  ray  of  light  is  transmitted 
without  double  refraction. 

Ordinary  light,  according  to  the  wave  theory,  is  due  to  vibrations  occur- 
ring in  all  planes  transversely  to  the  direction  of  the  propagation  of  the  wave. 
Light  is  said  to  be  plane  polarised  when  the  vibrations  take  place  all  in  one 
plane.  The  two  rays  produced  by  double  refraction  are  both  polarised,  one 
in  one  plane,  the  other  in  a  plane  at  right  angles  to  this  one.     Doubly  refract- 


APPENDIX 


223 


ing  bodies  are  called  anisotropoiis ;    singly  refracting  bodies,  isotrojJOiifi. 
The  effect  of  polarisation  may  be  very  roughly  illustrated  by  a  model. 

If  a  string  be  stretched  as  in  the  figure,  and  then  touched  with  the  finger, 
it  can  be  made  to  vibrate,  and  the  vibrations  will  be  free  to  occur  from  above 
down,  or  from  side  to  side,  or  in  any  intermediate  position.  If,  however,  a 
disc  with  a  vertical  slit  be  placed  on  the  course  of  the  string,  the  vibrations 
will  all  be  obliged  to  take  place  in  a  vertical  plane,  any  side  to  side  movement 
being  stopped  by  the  edges  of  the  slit  ^  (fig.  69). 


Light  can  be  polarised  not  only  by  the  action  of  crystals,  but  by  reflection 
from  a  surface  at  an  angle  which  varies  for  different  substances  (glass 
54°  35',  water  52°  45',  diamond  68°,  quartz  57°  32',  &c.).  It  is  also  found 
that  certain  non-crystalline  substances,  like  muscle,  cilia,  &c.,  are  doubly 
refracting. 

Nicol's  Prism  is  the  polariser  usually  employed  in  polariscopes ;  it  con- 
sists of  a  rhombohedron  of  Iceland  spar  divided  into  two  by  a  section 
through  its  obtuse  angles.  The  cut  surfaces  are  polished  and  cemented 
together  in  their  former  position  with  Canada  balsam.     By  this  means  the 


Fig.  70. 


ordinary  ray  is  totally  reflected  through  the  Canada  balsam  ;  the  extra- 
ordinary ray  passes  on  and  emerges  in  a  direction  parallel  to  the  entering 
ray.  In  this  polarised  ray  there  is  nothing  to  render  its  peculiar  condition 
visible  to  the  naked  eye ;  but  if  the  eye  is  aided  by  a  second  Nicol's  prism, 
which  is  called  the  analyser,  it  is  possible  to  detect  the  fact  that  it  is 
polarised. 

This  may  be  again  illustrated  by  reference  to  our  model  (fig.  70). 

Suppose  that  the  string  is  made  to  vibrate,  and  that  the  waves  travel  in 
the  direction  of  the  arrow.     From  the  fixed  point  c  to  the  disc  a,  the  string 

'  Such  a  model  is,  of  course,  imperfect ;  it  does  not,  for  instance,  represent  the 
splitting  of  the  ray  into  two,  and  moreover  the  polarisation  takes  place  on  each 
side  of  the  slit ;  whereas,  in  regard  to  light,  it  is  only  the  rays  on  one  side  of  a 
polariser,  viz.  those  that  have  passed  through  it,  which  are  polarised. 


224 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY. 


is  theoretically  free  to  vibrate  in  any  plane ;  ^  but  after  passing  through  the 
vertical  slit  in  a,  the  vibrations  must  all  be  vertical  also ;  if  a  second  similar 
disc  h  be  placed  further  on,  the  vibrations  will  also  pass  on  freely  to  the  other 
extremity  of  the  string  d,  if  as  in  the  figure  (fig.  70)  the  slit  in  b  be  also  placed 
vertically.  If,  however,  b  is  so  placed  that  its  slit  is  horizontal  (fig.  71)  the 
vibrations  will  be  extinguished  on  reaching  b,  and  the  string  between  b  and  d 
will  be  motionless. 

I 


Fig.  71. 

c  here  represents  a  source  of  light ;  the  vibrations  of  the  string  represent 
the  undulations  which  by  the  Nicol's  prism  a  are  polarised  so  as  to  occur  in 
one  plane  only  ;  if  the  second  Nicol's  prism  or  the  analyser  b  is  parallel  to  the 
first,  the  vibrations  will  pass  on  to  the  ej^e,  which  is  represented  by  d ;  but  if 
the  planes  of  the  two  nicols  are  at  right  angles,  the  vibrations  allowed  to  pass 
through  the  first  are  extinguished  by  the  second,  and  so  no  light  reaches  the  eye. 
In  intermediate  positions,  b  will  allow  only  some  of  the  light  to  pass  through 
it.  It  must  be  clearly  understood  that  a  Nicol's  prism  contains  no  actual  slits, 
but  the  arrangement  of  its  molecules  is  such  that  their  action  on  the  particles 
of  aether  may  be  compared  to  the  action  of  slits  in  a  diaphragm  to  vibratiojis 
of  more  tangible  materials  than  aether. 

The  Polarising  Microscope  consists  of  an  ordinary  microscope  with  certain 
additions;  below  the  stage  is  the  polarising  nicol ;  in  the  eye-piece  is  the 
analysing  nicol ;  the  eye-piece  is  so  arranged  that  it  can  be  rotated ;  thus  the 
directions  of  the  two  nicols  can  be  made  parallel,  and  then  the  field  is  bright ; 
or  crossed,  and  then  the  field  is  dark.  The  stage  of  the  microscope  is  arranged 
so  that  it  can  also  be  rotated. 

The  polarising  microscope  is  used  to  detect  doubly-refracting  substances. 
Let  the  two  nicols  be  crossed,  so  that  the  field  is  dark ;  interpose  between 
the  two,  that  is,  place  upon  the  stage  of  the  microscope  a  doubly-refracting 
plate  of  which  the  principal  plane  is  parallel  to  the  first  prism  or  polariser  ; 
the  ray  from  the  first  prism  is  unaffected  by  the  plate,  but  will  be  extinguished 
by  the  second ;  the  field  therefore  still  remains  dark.  If  the  plate  is  parallel 
to  the  second  nicol  the  field  is  also  dark  ;  but  in  any  intermediate  position 
the  light  will  be  transmitted  by  the  second  nicol.  In  other  words,  if  between 
two  crossed  nicols,  which  consequently  appear  dark,  a  substance  be  interposed 
which  in  certain  positions  causes  the  darkness  to  give  place  to  illumination, 
that  substance  is  doubly  refractive.  How  this  takes  place  may  be  explained 
as  follows : — 

Let  N^Nj  (fig.  72}  represent  the  direction  of  the  principal  plane  of  the 
first  nicol,  and  N0N2  that  of  the  second.     They  are  at  right  angles,  and  so 

»  The  imperfection  of  the  model  has  been  explained  in  the  preceding  footnote. 


APPENDIX 


225 


the  ray  transmitted  by  the  first  will  be  extinguished  by  the  second.     Let 

PP  represent  the  principal  plane  of  the  interposed  doubly -refractive  plate. 

The  extraordinary  ray  transmitted  by  NjNj  vibrates  in  the  plane  NjN,,  and 

falls  obliquely  on  the  plate  PP ;  it  is  by  this  plate  itself  split  into  two  rays, 

an  ordinary  and  an  extraordinary  one,  at  right  angles  to  one  another,  one 

vibrating  in  the  plane  PP,  the  other  in  the  plane  P^P^     These  two  rays 

meet  the  second  nicol,  which  can  only  transmit  vibrations  in  the  plane  NgNg. 

The  vibrations  in  PP  can  be  resolved 

into   a  vibration  in    NjNi    and   a 

vibration  in  N.^N., ;   the  former  is 

extinguished,  the  latter  transmitted. 

Similarly  the  vibration  in  P^P^  can 

be   resolved   into   two   sub-rays  in 

NjNj   and    N2N.J   respectively,   the 

latter  only  being  transmitted.     The 

illumination   is   thus   due    to    two 

sub-rays,  one  of  the  vibrations   in 

PP,  the  other  of  those  in  P^P^  which 

have  been  made  to  vibrate  in  N^N^. 

Now,  although  these  two  sub- 
rays  vibrate  in  the  same  plane, 
they  are  of  different  velocities ; 
hence  the  phases  of  the  vibrations 
do     not     coincide,    and    thus    the 

phenomena  of  interference  are  obtained.  If  we  have  two  sets  of  vibrations 
fused,  the  crest  of  one  wave  may  coincide  with  the  crest  of  the  other, 
in  this  case  the  wave  will  be  higher;  or  the  crest  of  one  may  coincide 
with  the  hollow  of  the  other,  that  is,  the  undulation  would  be  extinguished ; 
in  ether  intermediate  cases,  the  movement  would  be  interfered  with,  either 
helped  or  hindered,  more  or  less.  Interference  in  the  case  of  many  kinds 
of  doubly-refracting  substances  (Iceland  spar  is  in  this  an  exception)  shows 
itself  in  the  extinction  of  certain  rays  of  the  white  light,  and  the  light  seen 
through  the  second  nicol  is  white  light  minus  the  extinguished  rays  ;  those 
extinguished  and  those  -transmitted  will  together  form  white  light,  and  are 
thus  complementary.  Moreover,  the  rays  extinguished  in  one  position  of 
the  plate  will  be  transmitted  in  one  at  right  angles  and  vice  versa ;  thus  a 
crystal  showing  these  phenomena  of  pleochromatism,  as  it  is  termed,  will 
transmit  one  colour  in  one  position,  and  the  complementary  colour  in  a 
position  at  right  angles  to  the  first ;  blue  and  yellow,  and  red  and  green,  are 
the  pairs  of  colours  most  frequently  seen  in  this  way. 

Rotation  of  the  Plane  of  Polarisation. — Certain  crystals  such  as  those  of 
quartz,  and  certain  fluids  such  as  the  essence  of  turpentine,  aniseed,  &c., 
and  solutions  of  certain  substances  like  sugar  and  albumin,  have  the  power 
of  rotating  the  plane  of  polarised  light  to  the  right  or  left.  The  polarisation 
of  light  that  is  produced  by  a  quartz  crystal  is  different  from  that  produced 
by  a  rhombohedron  of  Iceland  spar.  The  light  that  passes  through  the 
latter  is  plane  polarised ;  the  light  that  passes  through  the  former  (quartz) 
is  circularly  polarised,  i.e.  the   two  sub -rays    are  made  up   of  vibrations 


226  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

which  occur  not  in  a  plane,  but  are  curved.  The  two  rays  are  circularly 
polarised  in  opposite  directions,  one  describing  circles  to  the  left,  the  other 
to  the  right ;  these  unite  on  issuing  from  the  quartz  plate  ;  and  the  net 
result  is  a  plane  polarised  ray  with  the  plane  rotated  to  right  or  left 
according  as  the  right  circularly  polarised  ray  or  the  left  proceeded  through 
the  quartz  with  the  greater  velocity.  There  are  two  kinds  of  quartz,  one 
which  rotates  the  plane  to  the  right  (dextro-rotatory),  the  other  to  the  left 
(IsBvo-rotatory). 

Gordon  explains  this  by  the  following  mechanical  illustration.  Ordinary 
light  may  be  represented  by  a  wheel  travelling  in  the  direction  of  its  axle, 
and  the  vibrations  composing  it  executed  along  any  or  all  of  its  spokes  (a). 
If  the  vibrations  all  take  place  in  the  same  direction,  i.e.  along  one  spoke, 
and  the  spoke  opposite  to  it  (b),  the  light  is  said  to  be  plane  polarised.  The 
two  spokes  as  they  travel  along  in  the  direction  of  the  arrow  will  trace  out 
a  plane  (see  fig.  73)  between  b  and  b'.     If  this  polarised  beam  be  made  to 


travel  now  through  a  solution  of  sugar,  the  net  result  is  that  the  plane  so 
traced  out  is  twisted  or  rotated ;  the  two  spokes,  as  in  bb',  do  not  trace  out 
a  plane,  but  we  must  consider  that  they  rotate  as  they  travel  along,  as 
though  guided  by  a  spiral  or  screw  thread  cut  on  the  axis,  so  that  after  a 
certain  distance  the  vibrations  take  place  as  in  b" ;  later  in  b'",  and  so  on. 
This  effect  on  polarised  light  is  due  to  the  molecules  in  solution,  and  the 
amount  of  rotation  will  depend  on  the  strength  of  the  solution,  and  on  the 
length  of  the  column  of  the  solution  through  which  the  light  passes,  or,  in 
the  case  of  a  quartz  plate,  on  its  thickness. 

If  a  plate  of  quartz  be  interposed  between  two  nicols,  the  light  will  not  be 
extinguished  in  any  position  of  the  prisms,  but  will  pass  through  various 
colours  as  rotation  is  continued.  The  rotation  produced  for  different  kinds 
of  Ught  being  different,  white  hght  is  split  into  its  various  constituent  colours  ; 
and  the  angle  of  rotation  that  causes  each  colour  to  disappear  is  constant  for 
a  given  thickness  of  quartz  plate,  being  least  for  the  red  and  greatest  for  the 
violet.  These  facts  are  made  use  of  in  the  construction  of  polarimeters. 
Polarimeters  are  instruments  for  determining  the  strength  of  solutions  of 
sugar,  albumin,  &c.,  by  the  direction  and  amount  of  rotation  they  produce 
on  the  plane  of  polarised  light.  They  are  often  called  saccharimeters,  as 
they  are  specially  useful  in  the  estimation  of  sugar. 

POLARIMETERS 

Soleil's  Saccbarimeter. — This  instrument  (see  fig.  74)  consists  of  a  Nicol's 
prism,  d,  called  the  polariser :  this  polarises  the  light  entering  it,  and  the 
polarised  beam  then  passes  through  a  quartz  plate  {b  in  fig.  74),  3-75  mm. 


APPENDIX  227 

thick,  one  half  of  which  {d  in  fig.  75)  is  made  out  of  dextro-rotatory,  the  other 
half  {g  in  fig.  75)  of  laBVO-rotatory  quartz. 

The  light  then  passes  through  the  tube  containing  the  solution  in  the 
position  of  the  dotted  line  in  fig.  74,  then  through  a  quartz  plate  cut  per- 
pendicularly to  its  axis  {q  in  fig.  75),  then  through  an  arrangement  called 
a  compensator  (r  in  fig.  75),  then  through  a  second  nicol  called  the  analyser, 
and  lastly  through  a  telescope  (L  in  fig.  75). 

The  compensator  consists  of  two  quartz  prisms  (KR',  fig.  75)  cut  perpen- 
dicularly to  the  axis,  but  of  contrary  rotation  to  the  plate  just  in  front  of 


Fig.  74. — Soleil's  saccharimeter. 

them.  These  are  wedge-shaped,  and  slide  over  each  other,  the  sharp  end  of 
one  being  over  the  blunt  end  of  the  other.  By  a  screw  the  wedges  may  be 
moved  from  each  other,  and  this  diminishes  the  thickness  of  quartz  inter- 
posed ;  if  moved  towards  each  other  the  amount  of  quartz  interposed  is 
increased. 

The  effect  of  the  quartz  plate  {d,  g)  next  to  the  polariser  {i  in  fig.  75) 
is  to  give  the  polarised  light  a  violet  tint  when  the  two  nicols  are  parallel  to 
each  other.  But  if  the  nicols  are  not  parallel,  or  if  the  plane  of  the  polarised 
light  has  been  rotated  by  a  solution  in  the  tube,  one  half  the  field  will  change 

f   7^ 


HBO -   - B- 


E 


EiiliiiY^^^ 


r  

11' 

Fig.  75.— Diagram  of  optical  arrangements  in  Soleil's  saceharimeter. 

in  colour  to  the  red  end,  the  other  to  the  violet  end  of  the  spectrum,  because 
the  two  halves  of  the  quartz  act  in  the  opposite  way. 

The  instrument  is  first  adjusted  with  the  compensator  at  zero,  and  the 
nicols  parallel,  so  that  the  whole  field  is  of  one  colour.  The  tube  containing 
the  solution  is  then  interposed ;  and  if  the  solution  is  optically  inactive  the 
field  is  still  uniformly  violet.  But  if  the  solution  is  dextro-rotatory  the  two 
halves  will  have  different  tints ;  a  certain  thickness  of  the  compensating 
quartz  plate  which  is  Isevo-rotatory  must  be  interposed  to  make  the  tint  of 
the  two  halves  of  the  field  equal  again  ;  the  thickness  so  interposed  can  be 
read  off  in  amounts  corresponding  to  degrees  of  a  circle  by  means  of  a  vernier 

q2 


228 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


and  scale  (E  in  fig.  76)  worked  by  the  screw  which  moves  the  compensator. 
If  the  solution  is  Isevo-rotatory,  the  screw  must  be  turned  in  the  opposite 
direction. 

Zeiss' s  polarimeter  is  in  principle  much  the  same  as  Soleil's ;  the  chief 
difference  is  that  the  rotation  produced  by  the  solution  is  corrected,  not  by  a 
quartz  compensator,  but  by  actually  rotating  the  analyser  in  the  same 
direction,  the  amount  of  rotation  being  directly  read  off  in  degrees  of  a 
circle. 

Laurent's  polarimeter  is  a  more  valuable  instrument.  Instead  of  using 
daylight,  or  the  light  of  a  lamp,  monochromatic  light  (a  sodium  flame  pro- 


FiG.  76. — Laurent's  polarimeter. 


duced  by  volatilising  common  salt  in  a  colourless  gas  flame)  is  employed  ; 
the  amount  of  rotation  varies  for  different  colours  ;  and  observations  are 
recorded  as  having  been  taken  with  light  corresponding  to  the  1)  or  sodium 
line  of  the  spectrum.  The  essentials  of  the  instrument  are,  as  before,  a 
polariser,  a  tube  for  the  solution,  and  an  analyser.  The  polarised  light 
before  passing  into  the  solution  traverses  a  quartz  plate,  which,  however, 
covers  only  half  the  field,  and  retards  the  rays  passing  through  it  by  half  a 
wave-length.     In  the  0°  position  the  two  halves  of  the  field  appear  equally 


APPENDIX 


2^9 


illuminated :  in  any  other  position,  or  if  rotation  has  been  produced  by  the 
solution  when  the  nicols  have  been  set  at  zero,  the  two  halves  appear  un- 
equally illuminated.  This  is  corrected  by  means  of  a  rotation  of  the  analyser 
that  can  be  measured  in  degrees  by  a  scale  attached  to  it. 

The  specific  rotatory  power  of  any  substance  is  the  amount  of  rotation  in 
degrees  of  a  circle  of  the  plane  of  polarised  light  produced  by  1  gramme  of 
the  substance  dissolved  in  1  c.c.  of  liquid  examined  in  a  column  1  deci- 
metre long. 

If  a      =  rotation  observed. 

w     =  weight  in  grammes  of  the  substance  per  cubic  centimetre. 
I       =  length  of  tube  in  decimetres. 

[a]n  =  specific    rotation   for  light  with  wave-length  corresponding  to 
the  D  line  (sodium  flame). 


Then[u]i> 


a 


In  this  formula  +  indicates  that  the  substance  is  dextro-rotatory,  -  that  it 
is  laevo-rotatory. 

If,  on  the  other  hand,  [a]^  is  known,  and  we  wish  to  find  the  value  of  w, 
then  " 

I  a  Id  X  Z 

THE    SPECTRO-POLARIMETER 

This  instrument  is  one  in  which  a  spectroscope  and  polarising  apparatus 
are  combined  for  the  purpose  of  determining  the  concentration  of  substances 


Fig.  77.— Spectro-polarimeter  of  v.  Fleischl. 


which  rotate  the  plane  of  polarised  light,  It  was  invented  by  E.  v.  Fleischl 
for  the  estimation  of  sugar  in  diabetic  urine.  Its  chief  advantage  is  that  no 
difficulty  arises  in  forming  a  judgment  as  to  the  identity  of  two  coloured 


230  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

surfaces,  as  in  Soleil's  saccharimeter,  or  of  two  shades  of  the  same  colour,  as 
in  Laurent's  instrument.  The  light  enters  at  the  right-hand  end  of  the 
instrument,  is  polarised  by  the  Nicol's  prism  b,  and  then  passes  through  two 
quartz  plates,  cc,  placed  horizontally  over  each  other.  One  of  these  plates  is 
dextro-,  the  other  laevo-rotatory,  and  they  are  of  such  a  thickness  (7*75  mm.) 
that  the  green  rays  between  the  E  and  b  lines  of  the  spectrum  are  circularly 
polarised  through  an  angle  of  90°,  the  one  set  passing  off  through  the  upper 
quartz  to  the  left,  the  other  through  the  lower  to  the  right.  The  light  then 
continues  through  a  long  tube,//,  which  contains  15  c.c.  of  the  solution  under 
examination.  It  then  passes  through  an  analysing  nicol  d,  and  finally 
through  a  direct-vision  spectroscope,  e.  On  looking  through  the  instrument, 
the  tube  //  being  empty  or  filled  with  water  or  some  other  optically  inert 
substance,  two  spectra  are  seen,  one  over  the  other,  but  each  shows  a  dark 
band  between  E  and  b  owing  to  the  extinction  of  these  rays  by  the  circular 
polarisation,  produced  by  the  quartz.  The  analyser  can  be  rotated :  a 
vernier,  g,  is  attached  to,  and  moves  with  it,  round  a  circular  disc  (seen  in 
section  at  h)  graduated  in  degrees.  The  two  bands  in  the  spectra  coincide 
when  the  zeros  of  vernier  and  scale  correspond.  If  now  the  tube  /  is  filled 
with  an  optically  active  substance  like  sugar,  the  bands  are  shifted,  one  to  the 
right,  the  other  to  the  left,  according  to  the  direction  of  rotation  of  the  sub- 
stance in  /.  The  rotation  is  corrected  by  rotating  the  analyser  into  such  a 
position  that  the  two  bands  exactly  coincide  once  more  as  to  vertical  position. 
The  number  of  degrees  through  which  it  is  thus  necessary  to  move  the 
analyser  measures  the  amount  of  rotation  produced  by  the  substance  in  /, 
and  is  a  measure  of  the  concentration  of  the  solution.  The  degrees  marked 
on  the  circular  scale  are  not  degrees  of  a  circle,  but  an  arbitrary  degree  of 
such  a  length  that  each  corresponds  to  1  per  cent,  of  sugar  in  the  given 
length  of  the  column  of  fluid  in  //  (177'2  mm.). 

RELATION   BETWEEN   CIRCULAR   POLARISATION   AND    CHEMICAL 
CONSTITUTION 

The  first  work  in  this  direction  was  performed  by  Pasteur,  and  it  was  his 
publications  on  this  subject  that  brought  him  into  prominence.  He  foimd 
that  racemic  acid,  which  is  optically  inactive,  can  be  decomposed  into  two 
isomerides,  one  of  which  is  common  tartaric  acid  which  is  dextro-rotatory,  and 
the  other  tartaric  acid  differing  from  the  common  variety  in  being  laevo- 
rotatory.  The  salts  of  tartaric  acid  usually  exhibit  hemihedral  faces,  while 
those  of  racemic  acid  are  holohedral.  Pasteur  found  that,  although  all  the 
tartrate  crystals  were  hemihedral,  the  hemihedral  faces  were  situated  on 
some  crystals  to  the  right,  and  on  others  to  the  left  hand  of  the  observer,  so 
that  one  formed,  as  it  were,  the  reflected  image  of  the  other.  These  crystals 
were  separated,  purified  by  recrystallisation,  and  those  which  exhibited 
dextro-hemihedry  possessed  dextro-rotatory  power,  while  the  others  were 
lavo-rotatory.  Pasteur  further  showed  that  if  the  mould  Pe7iicillium  glaucum 
is  grown  in  a  solution  of  racemic  acid,  dextro-tartaric  acid  first  disappears, 
and  the  laevo-acid  alone  remains.  The  subject  remained  in  this  condition  for 
many  years  ;  it  was,  however,  conjectured  that  proba.bly  there  is  some  mole- 
cular condition  corresponding  to  the  naked-eye  crystalline  appearances  which 


APPENDIX  231 

produces  the  opposite  optical  effects  of  various  substances.  What  this  mole- 
cular structure  is,  was  pointed  out  independently  by  two  observers — Le 
Bel  in  Paris,  and  Van  't  HofT  in  Holland — who  published  their  researches 
within  a  few  days  of  each  other.  They  pointed  out  that  all  optically  active 
bodies  contain  one  or  more  asymmetric  carbon  atoms,  i.e.  one  or  more  atoms 
of  carbon  connected  with  four  dissimilar  groups  of  atoms,  as  in  the  following 
examples : — 

C.Ha  CO.OH 

^  I 

H-(<^)— CH3  H— ©_0H 

CH^.OH  CH— CO.OH 

[Amyl  alcohol]  [Malic  acid] 

The  question,  however,  remained — do  all  substances  containing  such 
atoms  rotate  the  plane  of  polarised  light  ?  It  was  found  that  they  do  not ; 
this  is  explained  by  Le  Bel  by  supposing  that  these,  like  racemic  acid,  are 
compounds  of  two  molecules — one  dextro-,the  other  laevo-rotatory  ;  that  this 


/4 


Fig.  78. 


was  the  case  was  demonstrated  by  growing  moulds,  the  fermenting  action  of 
which  is  to  separate  the  two  molecules  in  question.  Then  the  other  question 
— how  is  it  that  two  isomerides,  which  in  chemical  characteristics,  in  graphic 
as  well  as  empirical  formulae,  are  precisely  alike,  differ  in  optical  properties? 
— is  explained  ingeniously  by  Van  't  Hoff.  He  compares  the  carbon  atom  to 
a  tetrahedron  with  its  four  dissimilar  groups.  A,  B,  C,  D,  at  the  four  corners. 
The  two  tetrahedra  represented  in  fig.  78  appear  at  first  sight  precisely  alike ; 
but  if  one  be  superimposed  on  the  other,  C  in  one  and  D  in  the  other  could 
never  be  made  to  coincide.  This  difference  cannot  be  represented  in  any 
other  graphic  manner,  and  probably  represents  the  difference  in  the  way  the 
atoms  are  grouped  in  the  molecule  of  right-  and  left-handed  substances 
respectively. 

MERCURIAL   AIR-PUMPS 

Pfluger's  Pump. — I  is  a  large  glass  bulb  filled  with  mercury  ;  from  its  lower 
end  a  straight  glass  tube,  7n,  about  3  feet  long,  extends,  which  is  connected 
by  an  india-rubber  tube,  n,  with  a  reservoir  of  mercury,  o,  which  can  be 
raised  or  lowered  as  required,  by  a  simple  mechanical  arrangement.  From 
the  upper  end  of  the  bulb,  Z,  a  vertical  tube  passes ;  above  the  stopcock,  7c, 
this  has  a  horizontal  branch,  which  can  be  closed  by  the  stopcock,  /.  The 
vertical  part  is  continued  into  the  bent  tube,  which  dips  under  mercury  in 
the  trough,  h.  A  stopcock,  j,  is  placed  on  the  course  of  this  tube.  Beyond/ 
the  horizontal  tube  leads  into  a  large  double  glass  bulb,  a  6 ;  a  mercurial 
gauge,  e,  and  a  drying-tube,  d,  filled  with  pieces  of  pumice-stone  moistened 
with   sulphuric   acid,  are  interposed,     a  is  called  the  blood-bulb,  and  the 


232 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


blood  is  brought  into  it  by  the  tube  c ;  the  gases,  as  they  come  ofif,  cause  the 
blood  to  froth,  and  the  bulb,  6,  is  called  the  froth-chamber,  as  it  intercepts 
the  froth,  preventing  it  from  passing  into  the  rest  of  the  apparatus. 

The  pump  is  used  in  the  following  way :  I  is  filled  with  mercury,  the 
level  in  I  and  o  being  the  same ;  k  is  closed  ;  o  is  then  lowered,  and  when  it 


Fio.  79.— Diagram  of  PflUger's  pump. 


is  30  inches  lower  than  the  stopcock,  li,  the  mercury  in  I  falls  also,  leaving 
that  bulb  empty  :  j  being  closed  and  /  open,  Ti  is  then  opened,  and  the  air  in 
a,  6,  d,  &c.,  rushes  into  the  Torricellianvacuum  in  Z ;  /  is  closed  and  j  opened  ; 
the  reservoir,  o,  is  raised  ;  the  mercury  in  I  rises  also,  pushing  the  air  before 
it,  and  it  bubbles  out  into  the  atmosphere  through  the  mercury  (the  tube,  h, 
is  not  at  this  stage  in  position).     When  I  is  full  of  mercury,  h  andy  are  once 


APPENDIX 


233 


more  closed  and  o  is  again  lowered ;  when  I  is  thus  rendered  once  more  a 
vacuum,  h  and  /  are  opened  and  more  of  the  air  remaining  in  a,  6,  d,  &c. 
rushes  into  the  vacuum  ;  /  is  closed,  j  is  opened,  and  this  air  is  expelled  as 
before.  The  process  is  repeated  as  often  as  is  necessary  to  make  a,  6,  d,  &c. 
as  complete  a  vacuum,  as  indicated  by  the  mercury  in  the  gauge,  e,  as  is 
obtainable. 

a  being  now  empty,  and  the  stopcock,  /,  closed,  blood  is  introduced  by  the 
tube  c  ;  it  froths  and  gives  forth  all  its  gases,  especially  if  heated  to  40-45°  C. 
In  the  case  of  serum,  acid  has  to  be  added  to  disengage  the  more  firmly 
combined  carbonic  acid.^  The  bulb,  I,  is  once  more  rendered  a  vacuum,  and 
k  and  /  are  opened,  j  being  closed.  The  gas  from  a  and  h  rushes  into  the 
bulb  Z,  being  dried  as  it  passes  through  d;  f'm  then  closed  and  j  opened ;  the 
reservoir  o  is  raised,  and  as  the  mercury  in  I  rises  simultaneously,  it  pushes 
the  gases  into  the  cylinder,  7i,  which  is  filled  with  mercury  and  inverted  over 
the  end  of  the  bent  tube.  This  gas  can  be  subsequently  analysed.  By 
alternately  raising  and  lowering  o,  and  regulating  the  stopcocks  in  the  manner 
already  described,  all  the  gas  from  the 
quantity  of  blood  used  can  be  ultimately 
expelled  into  h. 

A  good  grease  for  the  stopcocks  is  a 
mixture  of  two  parts  of  vaseline  to  one  of 
white  wax. 

Alvergniat's  pump  has  the  advantage 
over  Pfliiger's  of  fewer  connections,  and 
all  of  these  are  surrounded  by  mercury, 
which  effectually  prevents  leakage  ;  it  has 
the  disadvantage  of  a  rather  small  bulb 
in  place  of  I,  and  thus  it  is  more  labour  to 
obtain  a  vacuum. 

Leonard  Hill's  pump.—  This  is  a  simpler 
instrument,  and  is  sufiicient  for  most 
purposes.  It  consists  of  three  glass  bulbs 
(B.B.  in  fig.  80),  which  we  will  call  the 
blood  bulb  ;  this  is  closed  above  by  a 
piece  of  tubing  and  a  clip,  a ;  this  is 
connected  by  good  india-rubber  tubing  to 
another    bulb,    d.      Above    d^    however, 

there  is  a  stopcock  with  two  ways  cut  through  it :  one  by  means  of 
which  B.B.  and  d  may  be  connected,  as  in  the  figure ;  and  another  seen 
in  section,  which  unites  d  to  the  tube  e,  when  the  stopcock  is  turned  through 
a  right  angle.  In  intermediate  positions  the  stopcock  cuts  off  all  communi- 
cation from  d  to  all  parts  of  the  apparatus  above  it ;  d  is  connected  by  tubing 
to  a  receiver,  E,  which  can  be  raised  or  lowered  at  will.  At  first  the  whole 
apparatus  is  filled  with  mercury,  E  being  raised.  Then,  a  being  closed,  E  is 
lowered,  and  when  it  is  more  than  the  height  of  the  barometer  (30  inches) 
below  the  top  of  B.B.  the  mercury  falls  and  leaves  the  blood  bulb  empty ;  by 
lowering  E  still  further,  d  can  also  be  rendered  a  vacuum.     A  few  drops  of 

'  Phosphoric  acid  is  usually  employed. 


^^3^ 


B.B 


Fig.  80. — L.  Hill's  air-pump. 


234 


ESSENTIALS  OF  CHEMICAL   PHYSIOLOGY 


mercury  should  be  left  behind  in  B.B.  B.B.  is  then  detached  from  the  rest 
of  the  apparatus  and  weighed,  the  clips,  a  and  6,  being  tightly  closed.  Blood 
is  then  introduced  into  it  by  connecting  the  tube  with  the  clip  a  on  it  to  ^ 
cannula  filled  with  blood  inserted  in  an  artery  or  vein  of  a  living  animal. 
Enough  blood  is  withdrawn  to  fill  about  half  of  one  of  the  bulbs.  This  is 
defibrinated  by  |^ shaking  it  with  a  few  drops  of  mercury  left  in  the  bulb. 
It  is  then  weighed  again  ;  the  increase  of  weight  gives  the  amount  of  blood 
which  is  being  investigated.  B.B.  is  then  once  more  attached  to  the  rest  of 
the  apparatus,  hanging  downwards,  as  in  the  side  drawing  in  fig.  80,  and 

the  blood  gases  boiled  off;  these  pass  into 
d,  which  has  been  made  a  vacuum ;  and 
then,  by  raising  K  again,  the  mercury 
rises  in  d,  pushing  the  gases  in  front  of 
it  through  the  tube  e  (the  stopcock  being 
turned  in  the  proper  direction)  into  the 
eudiometer  E,  which  has  been  filled  with, 
and  placed  over,  mercury.  The  gas  can 
then  be  measured  and  analysed. 

ANALYSIS    OF    GASES 

'  Waller's  -modification  of  Zuntz's  more 
complete  apparatus  will  be  found  very 
useful  in  performing  gas  analysis,  say  of 
the  expired  air  or  blood  gases  :  a  100  c.c. 
measuring  tube  graduated  in  tenths  of  a 
cubic  centimetre  between  75  and  100,  a 
filling  bulb  and  two  gas  pipettes  are  con- 
nected up  as  in  the  diagram. 

It  is  first  charged  with  acidulated 
water  up  to  the  zero  mark  by  raising 
the  filling  bulb  A,  tap  1  being  open.  It 
is  then  filled  with  100  c.c.  of  expired 
air,  the  filling  bulb  being  lowered  till 
the  fluid  in  the  tube  has  fallen  to  the 
100  mark.  Tap  1  is  now  closed.  The 
amount  of  carbonic  acid  in  the  expired 
air  is  next  ascertained  ;  tap  2  is  opened, 
and  the  air  is  expelled  into  the  gas  pipette 
B,  containing  strong  caustic  potash  solution,  by  raising  the  filHng  bulb  until 
the  fluid  has  risen  to  the  zero  mark  of  the  measuring  tube.  Tap  2  is  closed, 
and  the  air  left  in  the  gas  pipette  for  a  few  minutes,  during  which  the 
carbonic  acid  is  absorbed  by  the  potash.  Tap  2  is  then  opened  and  the  air 
drawn  back  into  the  measuring  tube  by  lowering  the  filling  bulb.  The 
volume  of  air  {minus  the  carbonic  acid)  is  read,  the  filling  bulb  being 
adjusted  so  that  its  contents  are  at  the  same  level  as  the  fluid  in  the 
measuring  tube.  The  amount  of  oxygen  is  next  ascertained  in  a  precisely 
similar  manner  by  sending  the  air  into  the  other  gas  pipette,  which  contains 
sticks  of  phosphorus  in  water,  and  measuring  the  loss  of  volume  (due  to 


Fig.  81.— Waller's  apparatus  for  gas 
analysis. 


APPENDIX  235 

absorption    of  oxygen)  in  the  air  when  drawn  back  into   the  tube.     The 
remaining  gas  is  nitrogen. 

KJELDAHL'S    METHOD    OF    ESTIMATING    NITROGEN 

This  simple  method  can  be  used  in  connection  with  most  substances  of 
physiological  importance.  Briefly,  it  consists  in  converting  all  the  nitrogen 
present  into  ammonia  by  means  of  sulphuric  acid  ;  then  rendering  alkaline 
with  soda,  and  distilling  over  the  ammonia  into  standard  acid,  the  diminu- 
tion in  acidity  of  which  measures  the  amount  of  ammonia  present. 

The  following  modification  of  the  original  method  is  used  in  this 
laboratory. 

About  1  gramme  of  the  substance  under  investigation  (or  in  the  case  of 
urine  when  one  wishes  to  make  an  estimation  of  total  nitrogen,  5  or  10  c.c. 
of  that  fluid)  is  placed  in  a  round  bottomed  Jena  flask  of  about  250  c.c. 
capacity,  and  20  c.c.  of  pure  sulphuric  acid  added.  Six  grammes  of 
potassium  sulphate  and  about  half  a  gramme  of  copper  sulphate  are  also 
added.  The  flask  should  be  provided  with  a  loose  balloon  stopper,  and 
arranged  in  a  sloping  direction  over  a  small  flame.  The  mixture  is  heated 
slowly  until  it  boils.  In  about  twenty  minutes  the  fluid  becomes  nearly 
colourless  ;  boiling  is  continued  for  another  forty-five  minutes.  By  this 
time  all  the  nitrogen  will  be  in  combination  as  ammonia. 

After  cooling,  the  fluid  is  washed  into  a  litre  flask  of  Jena  glass  (fig.  82,  A) 
and  water  added  until  the  total  volume  of  the  fluid  is  about  400  c.c.     Add 
then   an   excess  of   40   per   cent,  caustic   soda   solution, 
a  few  pieces  of  granulated  zinc  to  avoid  bumping  in  the 
subsequent    distillation,    and   immediately    fit   the    glass 
tube  B  into  the  neck  of   the  flask  by  means  of  a  well- 
fitting  rubber  stopper.     The  other    end  of  B  leads  into 
the  flask  C  which  contains  a  measured  amount  (50  or 
100  c.c.)  of  standard  sulphuric  acid  ;  ^  normal  acid  is  a 
convenient  strength  to  use.      The  bulb  D  shown  in  the 
figure  guards  against  regurgitation,  and  the  end  of  the 
tube  should  dip  just  below  the  surface  of  the  acid  in  C.      fig.  82.  —  Kjeidabi's 
The  mixture  in  the  flask  is  now  boiled  for  about  half  an  ^ppamtus*^''*'"'"^ 

hour  when    all   the  anmionia    will   have    distilled  over ; 
the  use  of  a  condenser  around  the  tube  B  is  unnecessary.     The  acidity  of  the 
standard  acid  is  then  determined  by  titrating  with  standard  alkali,  a  few 
drops  of  methyl  orange  being  added  to  act  as  the  indicator  of  the  end  of  the 
reaction  (this  gives  a  pink  colour  with  acid,  yellow  with  alkali). 

Example. — Suppose  1  gramme  of  a  nitrogenous  substance  is  taken,  and 
the  ammonia  distilled  over  into  100  c.c.  of  ^  normal  sulphuric  acid  ( =  20 
c.c.  normal  acid).  This  is  then  titrated  with  a  corresponding  solution  of 
soda,  and  it  is  found  that  the  neutral  point  is  reached  when  60  c.c.  of  the 
soda  solution  have  been  added.  The  other  40  c.c.  must  therefore  have  been 
neutralised  by  the  ammonia  derived  from  the  substance  under  investigation. 
This  40  c.c.  of  acid  =  8  c.c.  of  normal  acid  =  8  c.c.  of  normal  ammonia  = 
8  X  0'017  =  0*136  gramme  of  ammonia.  One  gramme  of  the  substance  ana- 
lysed, therefore,  yields  0-136  gramme  of  ammonia,  and  this  contains  0"112 


236  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

gramme  of  nitrogen  ;  100  grammes  will  therefore  contain  11-2  grammes  of 
nitrogen.  If  the  strength  of  the  acid  is  that  just  recommended,  each  c.c. 
corresponds  to  0'0028092  of  nitrogen. 

SOLUTIONS.     DIFFUSION.     DIALYSIS.     OSMOSIS. 

The  investigations  of  physical  chemists  dming  recent  years  have  given  us 
new  conceptions  of  the  meaning  of  the  words  that  stand  at  the  head  of  this 
article.  I  propose  to  state  what  these  new  conceptions  are,  and  briefly  to 
indicate  the  bearing  they  have  on  the  elucidation  of  physiological  problems. 

Solutions. — Water  is  the  fluid  in  which  soluble  materials  are  usually 
dissolved,  and  at  ordinary  temperatures  it  is  a  fluid,  the  molecules  of 
which  are  in  constant  movement ;  the  hotter  the  water  the  more  active  are 
the  movements  of  its  molecules,  until,  when  at  last  it  is  converted  into 
steam,  the  molecular  movements  become  much  more  energetic.  Perfectly 
pure  water  consists  of  molecules  with  the  formula  H2O,  and  these  molecules 
undergo  practically  no  dissociation  into  their  constituent  atoms,  and  it  is 
for  this  reason  that  pure  water  is  not  a  conductor  of  electricity. 

If  a  substance  like  sugar  is  dissolved  in  the  water,  the  solution  still 
remains  incapable  of  conducting  an  electrical  current.  The  sugar  molecules 
in  solution  are  still  sugar  molecules ;  they  do  not  undergo  dissociation. 

But  if  a  substance  like  salt  is  dissolved  in  the  water,  the  solution  is  then 
capable  of  conducting  electrical  currents,  and  the  same  is  true  for  most  acids, 
bases,  and  salts.  These  substances  do  undergo  dissociation,  and  the  simpler 
materials  into  which  they  are  broken  up  in  the  water  are  called  ions.  Thus 
if  sodium  chloride  is  dissolved  in  water,  a  certain  number  of  its  molecules 
become  dissociated  into  sodium  ions,  which  are  charged  with  positive  elec- 
tricity, and  chlorine  ions,  which  are  charged  with  negative  electricity. 
Similarly  a  solution  of  hydrochloric  acid  in  water  contains  free  hydrogen 
ions  and  free  chlorine  ions.  Sulphuric  acid  is  decomposed  into  hydrogen 
ions  and  ions  of  SOj^.  The  term  ion  is  thus  not  equivalent  to  atom,  for  an 
ion  may  be  a  group  of  atoms,  like  SO4,  in  the  example  just  given. 

Further,  in  the  case  of  hydrochloric  acid,  the  negative  charge  of  the 
chlorine  ion  is  equal  to  the  positive  charge  of  the  hydrogen  ion  ;  but  in  the 
case  of  the  sulphuric  acid,  the  negative  charge  of  the  SO^  ion  is  equal  to  the 
positive  charge  of  tv/o  hydrogen  ions.  We  can  thus  speak  of  monovalent, 
divalent,  trivalent,  &c.  ions. 

Ions  charged  with  positive  electricity  are  called  Teat-ions  because  they 
move  towards  the  kathode  or  negative  pole  ;  those  which  are  charged  with 
negative  electricity  are  called  an-ions  because  they  move  towards  the  anode 
or  positive  pole.     The  following  are  some  examples  of  each  class  : — 

Kat-ions.  Monovalent: — H,  Na,  K,  NH„  &c. 

Divalent :  Ca,  Ba,  Fe  (in  ferrous  salts),  &c. 

Trivalent:  Al,  Bi,  Sb,  Fe  (in  ferric  salts),  &c. 
An-ions.    Monovalent :  CI,  Br,  I,  OH,  NO3,  &c. 

Divalent : — S,  Se,  So^,  &c. 

Roughly  speaking,  the  greater  the  dilution  the  more  nearly  complete  is 
the  dissociation,  and  in  a  very  dilute  solution  of  such  a  substance  as  sodium 


APPENDIX  237 

chloride  we  may  consider  that  the  number  of  ions  is  double  the  number  of 
molecules  of  the  salt  present. 

The  ions  liberated  by  the  act  of  dissociation  are,  as  we  have  seen,  charged 
with  electricity,  and  when  an  electrical  current  is  led  into  such  a  solution 
it  is  conducted  through  the  solution  by  the  movement  of  the  ions.  Sub- 
stances which  exhibit  the  property  of  dissociation  are  known  as  electrolytes. 

The  conception  of  electrolytes,  which  we  owe  to  Arrhenius,  is  extremely 
important  in  view  of  the  question  of  osmotic  pressure  which  we  shall  be  con- 
sidering immediately ;  because  the  act  of  dissociation  increases  the  number 
of  particles  moving  in  the  solution  and  so  increases  the  osmotic  pressure,  for 
in  this  relation  the  ion  plays  the  same  part  as  a  molecule. 

The  liquids  of  the  body  contain  electrolytes  in  solution,  and  it  is  owing 
to  this  fact  that  they  are  able  to  conduct  electrical  currents. 

Another  physiological  aspect  of  the  subject  is  seen  in  a  study  of  the 
action  of  mineral  salts  in  solution  on  living  organisms  and  parts  of  organisms. 
Many  years  ago  Einger  showed  that  contractile  tissues  (heart,  cilia,  &c.) 
continue  to  manifest  their  activity  in  certain  saline  solutions.  Indeed,  as 
Howell  puts  it,  the  cause  of  such  rhythmical  action  is  the  presence  of  these 
inorganic  substances  in  the  blood  or  lymph  which  usually  bathes  them.  In 
the  case  of  the  heart,  the  sinus,  or  venous  end  of  the  heart,  is  peculiarly 
susceptible  to  the  stimulus  of  the  inorganic  salts,  and  the  rhythmical 
peristaltic  waves  so  started  travel  thence  over  the  rest  of  the  heart  muscle. 

Loeb  and  his  fellow  workers  have  confirmed  these  statements,  but  interpret 
them  now  as  ionic  action.  Contractile  tissues  will  not  contract  in  piu-e 
solutions  of  non-electrolytes  (like  sugar,  urea,  albumen).  But  different  con- 
tractile tissues  differ  in  the  nature  of  the  ions  which  are  most  favourable 
stimuli.  Thus  cardiac  muscle,  cilia,  amoeboid  movement,  karyokinesis,  cell 
division  are  all  alike  in  requiring  a  proper  adjustment  of  ions  in  their  sur- 
roundings if  they  are  to  continue  to  act,  but  the  proportions  must  be  different 
in  individual  cases.  Ions  affecting  the  rhythmical  contractions  may  be 
divided  into  three  classes  :  (1)  Those  which  produce  such  contractions  ;  of 
these  the  most  efficacious  is  Na.  (2)  Those  which  retard  rhythmical  con- 
tractions  ;  for  instance,  Ca  and  K.  (3)  Those  which  act  catalytically,  that 
is,  they  accelerate  the  action  of  Na,  though  they  do  not  themselves  produce 
rhythmical  contractions  directly  :  for  instance,  H  and  OH.  In  spite  of  the 
antagonistic  effect  of  Ca,  a  certain  amount  of  it  must  be  present  if  con- 
tractions are  to  continue  for  any  length  of  time.  Ions  produce  rhythmical 
contraction  only  because  they  affect  either  the  physical  condition  of  the 
colloidal  substances  (protein,  &c.)  in  protoplasm,  or  the  rapidity  of  chemical 
processes. 

Loeb  has  even  gone  so  far  as  to  consider  that  the  process  of  fertilisation 
is  mainly  ionic  action.  He  denies  that  the  nuclein  in  the  head  of  the  sperma- 
tozoon is  essential,  but  asserts  that  all  the  spermatozoon  does  is  to  act  as  the 
stimulus  in  the  due  adjustment  of  the  proportions  of  the  surrounding  ions. 
He  supports  this  view  by  numerous  experiments  on  ova,  in  which,  without 
the  presence  of  spermatozoa,  he  has  produced  larvae  (generally  imperfect 
ones,  it  is  true)  by  merely  altering  the  saline  constituents  of  the  fluid  that 
bathes  them.     Whether  such  a  notion  will  stand  the  test  of  further  verifica- 


238  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

tion  must  be  left  to  the  future.  So  also  must  the  equally  important  idea 
that  the  basis  of  a  nervous  impulse  is  electrolytic  action,  though  it  receives 
support  from  Macdonald's  recent  investigations. 

Gramme-molecular  Solutions. — From  the  point  of  view  of  osmotic  pressure 
a  convenient  unit  is  the  gramme-molecule.  A  gramme-molecule  of  any 
substance  is  the  quantity  in  grammes  of  that  substance  equal  to  its  molecular 
weight.  A  gramme -molecular  solution  is  one  which  contains  a  gramme - 
molecule  of  the  substance  per  litre.  Thus  a  gramme-molecular  solution  of 
sodium  chloride  is  one  which  contains  58*5  grammes  of  sodium  chloride 
(Na  =  23*05  ;  CI  =  35"45)  in  a  litre.  A  gramme-molecular  solution  of  grape 
sugar  (C^jHioO^j)  is  one  which  contains  180  grammes  of  grape  sugar  in  a 
litre.  A  gramme-molecule  of  hydrogen  (H^)  is  2  grammes  by  weight  of 
hydrogen,  and  if  this  were  compressed  to  the  volume  of  a  litre  it  would  be 
comparable  to  a  gramme -molecular  solution.  It  therefore  follows  that  a 
litre  containing  2  grammes  of  hydrogen  contains  the  same  number  of 
molecules  of  hydrogen  in  it,  as  a  litre  of  a  solution  containing  58*5  grammes 
of  sodium  chloride,  or  one  containing  180  grammes  of  grape  sugar  has  in 
it  of  salt  or  sugar  molecules  respectively.  To  put  it  another  way,  the  heavier 
the  weight  of  a  molecule  of  any  substance  the  more  of  that  substance  must 
be  dissolved  in  the  litre  to  obtain  its  gramme -molecular  solution.  Or  still 
another  way  :  if  solutions  of  various  substances  are  made  all  of  the  same 
strength  per  cent.,  the  solutions  of  the  materials  of  small  molecular  weight 
will  contain  more  molecules  of  those  materials,  than  the  solutions  of  the 
materials  which  have  heavy  molecules.  We  shall  see  that  the  calculation  of 
osmotic  pressure  depends  on  these  facts. 

Diifusion,  Dialysis,  Osmosis. — If  two  gases  are  brought  together  within  a 
closed  space,  a  homogeneous  mixture  of  the  two  is  soon  obtained.  This  is 
due  to  the  movements  of  the  gaseous  molecules  within  the  confining  space 
and  the  process  is  called  diffusion.  In  a  similar  way  diffusion  will  effect  in 
time  a  homogeneous  mixture  of  two  liquids  or  solutions.  If  v/ater  is  carefully 
poured  on  to  the  surface  of  a  solution  of  salt,  the  salt  or  its  ions  will  soon  be 
equally  distributed  throughout  the  whole.  If  a  solution  of  albumin  or  any 
other  colloidal  substance  is  used  instead  of  salt  in  the  experiment,  diffusion 
will  be  found  to  occur  much  more  slowly.  If,  instead  of  pouring  the  water 
on  to  the  surface  of  a  solution  of  salt  or  sugar,  the  two  are  separated  by  a 
membrane  made  of  such  a  material  as  parchment  paper,  a  similar  diffusion 
will  occur,  though  more  slowly  than  in  cases  where  the  membrane  is  absent. 
In  time,  the  water  on  each  side  of  the  membrane  will  contain  the  same 
quantity  of  sugar  or  salt.  Substances  which  pass  through  such  membranes 
are  called  crystalloids.  Substances  which  have  such  heavy  molecules 
(starch,  protein,  &c.)  that  they  will  not  pass  through  such  membranes  are 
called  colloids.  Diffusion  of  substances  in  solution  in  which  we  have  to  deal 
with  an  intervening  membrane  is  usually  called  dialysis.  The  process  of 
filtration  {i.e.  the  passage  of  materials  through  the  pores  of  a  membrane 
under  the  influence  of  mechanical  pressure)  may  be  excluded  in  such  experi- 
ments by  placing  the  membrane  (M)  vertically  as  shown  in  the  diagram 
(fig.  83),  and  the  two  fluids  A  and  B  on  each  side  of  it.  Diffusion  through  a 
membrane  is  not  limited  to  the  molecules  of  water,  but  it  may  occur  also  in 


APPENDIX 


239 


H 


A 

B 

:=rz^- 



Fig.  83. 


the  molecules  of  certain  substances  dissolved  in  the  water.  But  very  few  or 
no  membranes  are  equally  permeable  to  water  and  to  molecules  of  the 
substances  dissolved  in  the  water.  If  in  the  accompany- 
ing diagram  the  compartment  A  is  filled  with  pure  water, 
and  B  with  a  sodium  chloride  solution,  the  liquids  in 
the  two  compartments  will  ultimately  be  found  to  be 
equal  in  bulk  as  they  were  at  the  start,  and  each  will 
be  a  solution  of  salt  of  half  the  original  strength  of 
that  in  the  compartment  jB,  But  at  first  the  volume 
of  the  liquid  in  compartment  B  increases,  because  more 
water  molecules  pass  into  it  from  A  than  salt  mole- 
cules pass  from  B  into^.  The  term  osmosis  is  generally 
limited  to  the  stream  of  water  molecules  passing 
through  a  membrane,  while  the  term  dialysis  is  applied 
to  the  passage  of  the  molecules  in  solution  in  the 
water.  The  osmotic  stream  of  water  is  especially 
important,  and  in  connection  with  this  it  is  necessary 
to  explain  the  term  osmotic  pressure.     At  first,  then, 

osmosis  (the  diffusion  of  water)  is  more  rapid  than  the  dialysis  (the  diffusion 
of  the  salt  molecules  or  ions).  The  older  explanation  of  this  was  that  salt 
attracted  the  water,  but  we  now  express  the  fact  differently  by  saying  that 
the  salt  in  solution  exerts  a  certain  osmotic  pressure  :  the  result  of  the 
osmotic  pressure  is  that  more  water  flows  from  the  water  side  to  the  side  of 
the  solution  than  in  the  contrary  direction.  The  osmotic  pressure  varies 
with  the  amount  of  substance  in  solution,  and  is  also  altered  by  variations  of 
temperature,  occurring  more  rapidly  at  high  than  at  low  temperatures. 

If  we  imagine  two  masses  of  water  separated  by  a  permeable  membrane, 
as  many  water  molecules  will  pass  through  from  one  side  as  from  the  other, 
and  so  the  volumes  of  the  two  masses  of  water  will  remain  unchanged.     If 
now  we  imagine  the  membrane  M  is  not  permeable  except  to  water,  and  the 
compartment  A  contains  water,  and  the  compartment  B  contains  a  solution 
of   salt  or  sugar;  in  these  circumstances  water  will  pass  through   into   B, 
and  the  volume  of  B  will  increase  in  proportion  to  the  osmotic  pressure  of 
the  sugar  or  salt  in  solution  in  5,  but  no  molecules  of  sugar  or  salt  can  get 
through  into  A  from  B,  so  the  volume  of  fluid  in  A  will  continue  to  decrease, 
until  at  last  a  limit  is  reached.     The  determination  of  this  limit,  as  measured 
by  the  height  of  a  column  of  fluid  or  mercury  which  it  will  support,  will  give 
us  a  measurement  of  the  osmotic  pressure.     Membranes  of  this  nature  are 
called  semi-permeable.     One  of  the  best  kinds  of  semi-permeable  membrane  is 
ferrocyanide   of  copper.     This   may   be   made   by   taking  a  cell  of  porous 
earthenware  and  washing  it  out  first  with  copper  sulphate  and  then  with 
potassium  ferrocyanide.     An  insoluble  precipitate  of  copper  ferrocyanide  is 
thus  deposited  in  the  pores  of  the  earthenware.     If  such  a  cell  is  fiUed  with 
a  1-per-cent.  solution  of  sodium  chloride,  water  diffuses  in  till  the  pressure 
registered  by  a  manometer  connected  to  it  registers  the  enormous  height  of 
5,000  millimetres  of  mercury.     Theoretically  it  is  possible  to  measure  osmotic 
pressure  by  a  manometer  in  this  way,  but  practically  it  is  seldom  done,  and 
some  of  the   indirect   methods   of  measurement   described  later   are  used 


240  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

instead.  The  reason  for  this  is  that  it  has  been  found  difficult  to  construct 
a  membrane  which  is  absohitely  semi-permeable. 

Many  explanations  of  the  nature  of  osmotic  pressure  have  been  brought 
forward,  but  none  is  perfectly  satisfactory.  The  following  simple  explanation 
is  perhaps  the  best,  and  may  be  rendered  most  intelligible  by  an  illustration. 
Suppose  we  have  a  solution  of  sugar  separated  by  a  semi-permeable  mem- 
brane from  water :  that  is,  the  membrane  is  permeable  to  water  molecules, 
but  not  to  sugar  molecules.  The  streams  of  water  from  the  two  sides  will 
then  be  unequal ;  on  one  side  we  have  water  molecules  striking  against  the 
membrane  in  what  we  may  call  normal  numbers,  while  on  the  other  side 
both  water  molecules  and  sugar  molecules  are  striking  against  it.  On  this 
side,  therefore,  the  sugar  molecules  take  up  a  certain  amount  of  room,  and 
do  not  allow  the  water  molecules  to  get  to  the  membrane  ;  the  membrane  is, 
as  it  were,  screened  against  the  water  by  the  sugar,  therefore  fewer  water 
molecules  will  get  through  from  the  screened  to  the  unscreened  side  than 
vice  versa.  This  comes  to  the  same  thing  as  saying  that  the  osmotic  stream 
of  water  is  greater  from  the  unscreened  water  side  to  the  screened  sugar  side 
than  it  is  in  the  reverse  direction.  The  more  sugar  molecules  that  are 
present,  the  greater  will  be  their  screening  action,  and  thus  we  see  that  the 
osmotic  pressure  is  proportional  to  the  number  of  sugar  molecules  in  the 
solution :  that  is,  to  the  concentration  of  the  solution. 

Osmotic  pressure  is,  in  fact,  equal  to  that  which  the  dissolved  substance 
would  exert  if  it  occupied  the  same  space  in  the  form  of  a  gas  (Van  't  Hofif's 
hypothesis).  The  nature  of  the  substance  makes  no  difference  ;  it  is  only  the 
number  of  molecules  which  causes  osmotic  pressure  to  vary.  The  osmotic 
pressure,  however,  of  substances  like  sodium  chloride,  which  are  electrolytes,  is 
greater  than  what  one  would  expect  from  the  number  of  molecules  present. 
This  is  because  the  molecules  in  solution  are  split  into  their  constituent  ions, 
and  an  ion  plays  the  same  part  as  a  molecule,  in  questions  of  osmotic  pressure. 
In  dilute  solutions  of  sodium  chloride  ionisation  is  more  complete,  and  as  the 
total  number  of  ions  is  then  nearly  double  the  number  of  original  molecules, 
the  osmotic  pressure  is  nearly  double  what  would  have  been  calculated  from 
the  number  of  molecules. 

The  analogy  between  osmotic  pressure  and  the  partial  pressure  of  gases 
is  complete,  as  may  be  seen  from  the  following  statements  : — 

1.  At  a  constant  temperature  osmotic  pressure  is  proportional  to  the 
concentration  of  the  solution  (Boyle-Mariotte's  law  for  gases). 

2.  With  constant  concentration,  the  osmotic  pressure  rises  with  and  is 
proportional  to  the  temperature  (Gay-Lussac's  law  for  gases). 

3.  The  osmotic  pressure  of  a  solution  of  different  substances  is  equal  to 
the  sum  of  the  pressures  which  the  individual  substances  would  exert  if  they 
were  alone  in  the  solution  (Henry-Dalton's  law  for  partial  pressure  of  gases). 

4.  The  osmotic  pressure  is  independent  of  the  nature  of  the  substance  in 
solution,  and  depends  only  on  the  number  of  molecules  or  ions  in  solution 
(Avogadro's  law  for  gases). 

Calculation  of  Osmotic  Pressure.  —We  may  best  illustrate  this  by  an  example 
and  to  simplify  matters  we  will  take  an  example  in  the  case  of  a  non-electro- 
lyte like  sugar.     We  shall  then  not  have  to  take  into  account  any  electrolytic 


APPENDIX  241 

dissociation  of  the  molecules  into  ions.     We  will  suppose  we  want  to  calcu- 
late the  osmotic  pressure  of  a  1 -par-cent,  solution  of  cane  sugar. 

One  gramme  of  hydrogen  at  atmospheric  pressure  and  0"  C.  occupies  a 
volume  of  11'2  litres;  two  grammes  of  hydrogen  will  therefore  occupy  a 
volume  of  22*4  litres.  A  gramme-molecule  of  hydrogen — that  is,  2  grammes 
of  hydrogen — when  brought  to  the  volume  of  1  litre  will  exert  a  gas  pressure 
equal  to  that  of  22*4  litres  compressed  to  1  litre — that  is,  a  pressure  of  22*4 
atmospheres.  A  gramme-molecular  solution  of  cane  sugar,  since  it  contains 
the  same  number  of  molecules  in  a  litre,  must  therefore  exert  an  osmotic  pres- 
sure of  22*4  atmospheres  also.  A  gramme-molecular  solution  of  cane  sugar 
(CiaH^.jOn)  contains  342  grammes  of  cane  sugar  in  a  litre.  A  1-per-cent. 
solution  of  cane  sugar  contains  only  10  grammes  of  cane  sugar  in  a  litre  of 
water  ;  hence  the  osmotic  pressure  of  a  1-per-cent.  solution  of  cane  sugar  is 

-  X  22*4  atmospheres,  or  0*65    of   an   atmosphere,  which  in  terms  of  a 

column  of  mercury  =  760  x  0-65  =  494  mm. 

It  would  not  be  possible  to  make  such  a  calculation  in  the  case  of  an 
electrolyte,  because  we  should  not  know  how  many  molecules  had  been 
ionised.  In  the  liquids  of  the  body,  both  electrolytes  and  non-electrolytes 
are  present,  and  so  a  calculation  is  here  also  impossible. 

We  have  seen  the  difficulty  of  directly  measuring  osmotic  pressure  by  a 
manometer ;  we  now  see  that  mere  arithmetic  often  fails  us  ;  and  so  we  come 
to  the  question  to  which  we  have  been  leading  up,  viz.  how  osmotic  pressure 
is  actually  determined. 

Determination  of  Osmotic  Pressure  by  means  of  the  Freezing-point. — This  is 
the  method  which  is  almost  universally  employed.  A  very  simple  apparatus 
(Beckmann's  differential  thermometer)  is  all  that  is  necessary.  The  principle 
on  which  the  method  depends  is  the  following : — The  freezing-point  of  any 
substance  in  solution  in  water  is  lower  than  that  of  water  ;  the  lowering  of 
the  freezing-point  is  proportional  to  the  molecular  concentration  of  the  dis- 
solved substance,  and  that,  as  we  have  seen,  is  proportional  to  the  osmotic 
pressure. 

When  a  gramme-molecule  of  any  substance  is  dissolved  in  a  litre  of  water, 
the  freezing-point  is  lowered  by  1*87°  C,  and  the  osmotic  pressure  is,  as  we 
have  seen,  equal  to  22*4  atmospheres  :  that  is,  22*4  x  760  =  17,024  mm.  of 
mercury. 

We  can  therefore  calculate  the  osmotic  pressure  of  any  solution  if  we 
know  the  lowering  of  its  freezing-point  in  degrees  Centigrade  ;  the  lowering 
of  the  freezing-point  is  usually  expressed  by  the  Greek  letter  A. 

Osmotic  pressure  =  ,~-  x  17,024. 
1-87 

For  example,  a  1-per-cent.  solution  of  sugar  would  freeze  at   -0-052°  C. ; 

.    ,,        o       -052x17,024     .^o  , 

its  osmotic  pressure  is  therefore  t'-^^ —  =473  mm.,  a  number  approxi- 

1'87 

mately  equal  to  that  we  obtained  by  calculation. 

Mammalian  blood  serum  gives  A  =  0*56°  C.  A  0'9-per-cent.  solution  of 
sodium  chloride  has  the  same  A  ;  hence  serum  and  a  0-9-per-cent.  solution 
of  common  salt  have  the  same  osmotic  pressure,  or  are  isotonic.     The  osmotic 


242  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

'56  X  17  024 

pressure  of  blood  serum  is        -     _' — =5,000    mm.    of    mercury    approxi- 

1*87 

mately,  or  a  pressure  of  nearly  7  atmospheres. 

The  osmotic  pressure  of  solutions  may  also  be  compared  by  observing 
their  effect  on  red  corpuscles,  or  on  vegetable  cells  such  as  those  in  Trades- 
cantia.  If  the  solution  is  hypertonic^  i.e.  has  a  greater  osmotic  pressure 
than  the  cell  contents,  the  protoplasm  shrinks  and  loses  water,  or,  if  red 
corpuscles  are  used",  they  become  crenated.  If  the  solution  is  hypotonic,  e.g. 
has  a  smaller  osmotic  pressure  than  the  material  within  the  cell-wall,  no 
shrinking  of  the  protoplasm  in  the  vegetable  cell  occurs,  and  if  red  corpuscles 
are  used  they  swell  and  liberate  their  pigment.  Isotonic  solutions  produce 
neither  of  these  effects,  because  they  have  the  same  molecular  concentration 
and  osmotic  pressure  as  the  material  within  the  cell- wall. 

Physiological  Applications. — It  will  at  once  be  seen  how  important  all  these 
considerations  are  from  the  physiological  standpoint.  In  the  body  we  have 
aqueous  solutions  of  various  substances  separated  from  one  another  by 
membranes.  Thus  we  have  the  endothelial  walls  of  the  capillaries  separating 
the  blood  from  the  lymph  ;  we  have  the  epithelial  waUs  of  the  kidney  tubules 
separating  the  blood  and  lymph  from  the  urine ;  we  have  similar  epithelium 
in  all  secreting  glands ;  and  we  have  the  wall  of  the  alimentary  canal 
separating  the  digested  food  from  the  blood-vessels  and  lacteals.  In  such 
important  problems,  then,  as  lymph-formation,  the  formation  of  urine  and 
other  excretions  and  secretions,  and  absorption  of  food,  we  have  to  take  into 
account  the  laws  which  regulate  the  movements  both  of  water  and  of  sub- 
stances which  are  held  in  solution  by  the  water.  In  the  body  osmosis  is  not 
the  only  force  at  work,  but  we  have  also  to  consider  filtration  :  that  is,  the 
forcible  passage  of  materials  through  membranes,  due  to  differences  of 
mechanical  pressure.  Further  complicating  these  two  processes  we  have  to 
take  into  account  another  force  :  namely,  the  secretory  or  selective  activity  of 
the  living  cells  of  which  the  membranes  in  question  are  composed.  This  is 
sometimes  called  by  the  name  vital  action,  which  is  an  unsatisfactory  and 
unscientific  expression.  The  laws  which  regulate  filtration,  imbibition,  and 
osmosis  are  fairly  well  known  and  can  be  experimentally  verified.  But  we 
have  undoubtedly  some  other  force,  or  some  other  manifestation  of  force,  in 
the  case  of  Uving  membranes.  It  probably  is  some  physical  or  chemical 
property  of  living  matter  which  has  not  yet  been  brought  into  line  with  the 
known  chemical  and  physical  forces  which  operate  in  the  inorganic  world. 
We  cannot  deny  its  existence,  for  it  sometimes  operates  so  as  to  neutralise 
the  known  forces  of  osmosis  and  filtration. 

The  more  one  studies  the  question  of  lymph-formation,  the  more  con- 
vinced one  becomes  that  mere  osmosis  and  filtration  will  not  explain  it  entirely. 
The  basis  of  the  action  is  no  doubt  physical,  but  the  living  cells  do  not 
behave  like  the  dead  membranes  of  a  dialyser  ;  they  have  a  selective  action, 
picking  out  some  substances  and  passing  them  through  to  the  lymph,  while 
they  reject  others. 

The  question  of  gaseous  interchanges  in  the  lungs  has  been  another 
battlefield  of  a  similar  kind.  Some  maintain  that  all  can  be  explained 
by  the  laws  of  diffusion  of  gases ;  others  assert  that  the  action  is  wholly 


APPENDIX  248 

vital.  Probably  those  are  most  correct  who  admit  a  certain  amount  of  truth 
in  both  views ;  the  main  facts  are  explicable  on  a  physical  basis,  but  there 
are  also  some  puzzling  data  which  show  that  the  pulmonary  epithelium  is 
able  to  exercise  some  other  force  as  well,  which  interferes  to  some  extent  with 
the  known  physical  process.  Take  again  the  case  of  absorption.  The  object 
of  digestion  is  to  render  the  food  soluble  and  diffusible ;  it  can  hardly  be  sup- 
posed that  this  is  useless  ;  the  readily  diffusible  substances  will  pass  more 
easily  through  into  the  blood  and  lymph  :  but  still,  as  Waymouth  Keid  has 
shown,  if  the  living  epithelium  of  the  intestine  is  removed,  absorption  comes 
very  nearly  to  a  standstill,  although  from  the  purely  physical  standpoint 
removal  of  the  thick  columnar  epithelium  would  increase  the  faciUties  for 
osmosis  and  filtration. 

The  osmotic  pressure  exerted  by  crystalloids  is  very  considerable,  but 
their  ready  diffusibility  limits  their  influence  on  the  flow  of  water  in  the 
body.  Thus,  if  a  strong  solution  of  salt  is  injected  into  the  blood,  the  first 
effect  will  be  the  setting  up  of  an  osmotic  stream  from  the  tissues  to  the 
blood.  The  salt,  however,  would  soon  diffuse  out  into  the  tissues,  and  would 
now  exert  osmotic  pressure  in  the  opposite  direction.  Moreover,  both  effects 
will  be  but  temporary,  because  excess  of  salt  is  soon  got  rid  of  by  the 
excretions. 

Osmotic  Pressure  of  Proteins. — It  has  been  generally  assumed  that  proteins, 
the  most  abundant  and  important  constituents  of  the  blood,  exert  little  or  no 
osmotic  pressure.  Starling,  however,  has  claimed  that  they  have  a  small 
osmotic  pressure  ;  if  this  is  so,  it  is  of  importance,  for  proteins,  unlike  salt,  do 
not  diffuse  readily,  and  their  effect  therefore  remains  as  an  almost  permanent 
factor  in  the  blood.  Starling  gives  the  osmotic  pressure  of  the  proteins  of 
the  blood-plasma  as  equal  to  30  mm.  of  mercury.  By  others  this  is  attributed 
to  the  inorganic  salts  with  which  proteins  are  always  closely  associated. 
Moore,  for  instance,  finds  that  the  purer  a  protein  is,  the  less  is  its  osmotic 
pressure  ;  the  same  is  true  for  other  colloidal  substances.  It  really  does  not 
matter  much,  if  the  osmotic  force  exists,  whether  it  is  due  to  the  protein 
itself,  or  to  the  saline  constituents  which  are  almost  an  integral  part  of  a 
protein.  It  is  merely  interesting  from  the  theoretical  point  of  view.  We 
should  from  the  theoretical  standpoint  find  it  difficult  to  imagine  that  a  pure 
protein  can  exert  more  than  a  minimal  osmotic  pressure.  It  is  made  up  of 
such  huge  molecules  that,  even  when  the  proteins  are  present  to  the  extent 
of  7  or  8  per  cent.,  as  they  are  in  blood-plasma,  there  are  comparatively  few 
protein  molecules  in  solution,  and  probably  none  in  true  solution.  Still,  by 
means  of  this  weak  but  constant  pressure  it  is  possible  to  explain  the  fact  that 
an  isotonic  or  even  a  hypertonic  solution  of  a  diffusible  crystalloid  may  be 
completely  absorbed  from  the  peritoneal  cavity  into  the  blood. 

The  functional  activity  of  the  tissue  elements  is  accompanied  by  the 
breaking  down  of  their  protein  constituents  into  such  simple  materials  as  uvea 
(and  its  precursors),  sulphates,  and  phosphates.  These  materials  pass  into 
the  lymph,  and  increase  its  molecular  concentration  and  its  osmotic  pressure  ; 
thus  water  is  attra.cted  (to  use  the  older  way  of  putting  it)  from  the  blood  to 
the  lymph,  and  so  the  volume  of  the  lymph  rises  and  its  flow  increases.  On 
the  other  hand,  as  these  substances  accumulate  in  the  lymph  they  will  in 

r2 


244  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 

time  attain  there  a  greater  concentration  than  in  the  blood,  and  so  they  will 
diffuse  towards  the  blood,  by  which  they  are  carried  to  the  organs  of 
excretion. 

But,  again,  we  have  a  difficulty  with  the  proteins ;  they  are  most  important 
for  the  nutrition  of  the  tissues,  but  they  are  practically  indiffusible.  We 
must  provisionally  assume  that  their  presence  in  the  lymph  is  due  to  filtration 
from  the  blood.  The  plasma  in  the  capillaries  is  under  a  somewhat  higher 
pressure  than  the  lymph  in  the  tissues,  and  this  tends  to  squeeze  the 
constituents  of  the  blood,  including  the  proteins,  through  the  capillary  walls. 
I  have,  however,  already  indicated  that  the  question  of  lymph-formation  is 
one  of  the  many  physiological  problems  which  await  solution  by  the 
physiologists  of  the  future. 


INDEX 


In  cases  where  several  references  are  made  to  any  subject,  the  figure  in  heavy  type  indicates 
where  the  principal  matter  in  relation  to  the  subject  is  to  be  found. 


A 

Abkin,  123 

Absorption,  95,  243 ;  of  carbohydrates, 
96  ;  of  fats,  99 ;  of  proteins,  96 

Absorption  bands,  117,  118,  119 

Absorption  spectra  of  haemoglobin  and 
its  derivatives,  119,  189  ;  of  myohae- 
matin,  199 ;  of  urinary  pigments, 
215 

Accessories  of  food,  60 

Acetic  acid,  24,  25,  63 

Acetompmia,  88 

Acetone,  88,  168 ;  test  for,  168 

Acetyl,  25 

Achroo-dextrin,  21,  68,  179 

Acid-albumin,  29,  30,  48,  62,  75,  79,  81, 
172 

Acid  haematin,  188 

Acid  haematoporphyrin,  189,  215  ;  ab- 
sorption bands  of,  189,  215 

Acid,  lactic,  see  Lactic  acid 

Acid  sodium  phosphate,  144 

Acid  tide,  144 

Acids,  vegetable,  4,  144 

Acrolein,  22,  24 

Acute  yellow  atrophy  of  liver,  147 

Adamkiewicz's  reaction,  27^  34,  39 

Adenase,  161 

Adenine,  46,  159,  160,  161 

Adipose  tissue,  2,  23 

Aerobic  micro-organisms,  66 

Aerotonometer,  132 

Agglutinating  action,  124 ;  agglutinins, 
124 

Air,  expired  and  inspired,  126  ;  pumps, 
mercurial,  231 

Alanine,  31,  33 

Alanyl,  36 

Albumin,  4,  237  ;  action  of  acids  and 
alkalis  on,  29,  30 

Albumin,  acid-,  29,  30  ;  alkali-,  29 


Albumin  in  urine,  166  ;  estimation  of, 
166  ;  tests  for,  156 

Albuminate,  48 

Albuminoids,  43 

Albuminometer  of  Esbach,  166 

Albumins,  37,  41,  42,  77,  171 

Albumose,  75 

Albumoses  and  peptone,  separation  of, 
182  ;    tests  for,  182 

Alcapton,  169 

Alcaptonuria,  169 

Alcohol,  action  of,  on  proteins,  182 

Alcoholic  fermentation,  56 ;  of  milk, 
56 

Alcohols,  4,  14,  24,  25 

Aldehyde,  14,  24 

Aldoses,  15 

Aleurone  grains,  38 

AlkaU-albumin,  29,  30,  48,  81,  172, 
187 

Alkaline  hiematin,  188 

Alkaline  haematoporphyrin,  215 ;  ab- 
sorption bands  of,  189,  215 

Alkaline  tide,  144 

Alkaloids,  60 

AUoxuric  bases,  46 

Allyl  alcohol,  24,  231 

Aluminium,  8 

Alvergniat's  pump,  233 

Amboceptor,  124 

Amidulin,  20 

Amino-acetic  acid,  see  Glycine 

Amino-acids,  31,  81,  85,  86,  97 

Amino-caproic  acid,  31,  86 

Amino- ethyl-sulphonic  acid,  91 

Amino-iso-butyl-acetic  acid,  86 

Amino-oxy-purine,  159 

Amino-propionic  acid,  31 

Amino-purine,  159 

Amino-pyrotartaric  acid,  32 

Amino-succinamic  acid,  32 

Amino-succinic  acid,  32 


246 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


Ammo- valeric  acid,  32 

Ammonia,  34,  151 

Ammoniacal     odour    of    putrid    urine, 

146 
Ammonio-magnesium  phosphate,  156 
Ammonium  carbonate   and  carbamate 

as  urea  precursors,  150 
Ammonium  cyanate,  145 
Ammonium    sulphate,    action    of,    on 

proteins,  27,  28,  40,  165,  182 
Ammonium  urate,  165 
Amoeboid  movements,  237 
Amylolytic  ferments,  65,  68,  184 
Amylopsin,  19,  65,  78,  80 ;    action  of, 

82 
Amyloses,  16,  65 
Anaerobic  micro-organisms,  66 
Analyses  of  gases,  234 
Animal  starch,  21 
Anions,  236 

Anisotropous  bodies,  223 
Anti-bodies,  77 
Antimony,  8 
Antipepsin,  77,  105 
Antiseptics,  63 
Antithrombin,  105, 107 
Antitoxic  material,  121 
Antitoxin,  123 
Antitrypsin,  77,  105 
Antivenin,  123 
Apparatus     necessary      for     practical 

work,  4,  5,  6 
Appendix,  217 

Aqueous  vapour,  tension  of,  8 
Arabinose,  178 
Arginase,  150,  161 
Arginine,  32,  33,  35,  41,  81,  150,  161 
Aromatic  amino-acids,  33 
Aromatic  series,  18 
Arsenic,  8 

Arterial  blood,  gases  of,  127 
Artificial  gastric  juice,  62 
Asparagine,  32 
Aspartic  acid,  32,  81 
Asphyxia,  128 
Atmosphere,  126 
Atomic  weights,  8 
Attraction  sphere,  2 
Atypical  globulin,  198 
Avogadro's  law,  127,  240 

B 

Bacilli,  65 

Bacteria,  65 

Bacterial  action,  85 

Bacterio-lysin,  122 

Barcroft's     apparatus     for     obtaining 

blood  gases,  135 
Barfoed's  reagent,  178 
Barium,  8 


Bath,  warm,  29 

Baumann  and  Wolkow  on  alcapton, 
169 

Bausteine,  98 

Beaumont,  Dr.,  on  gastric  secretion, 
70 

Beef,  57  ;  beef-tea,  59,  200 

Beetroot,  18 

Benger's  liquor  pancreaticus,  78,  186 ; 
liquor  pepticus,  62 

Benzene,  33 

Benzoic  acid,  162 

Bernard,  Claude,  on  glycogen  of  liver, 
96  ;  diabetic  puncture,  88 

Bile,  78,  89,  100  ;  amount  secreted,  90 
secretion  of,  89 ;  circulation,  90 
characters  of,  90 ;  constituents,  90 
mucin  of,  91 ;  pigments,  79,  90,  95 
salts  of,  79,  91,  94 ;  uses  of,  93 
actions  of,  94  ;  in  urine,  169 

Biliary  fistula,  89 

Bilirubin,  90,  91,  92,  93,  95,  213;  of 
meconium,  95 

Biliverdin,  91,  92,  95 

Bismuth,  8 

Biuret,  39,  77,  141;  reaction,  27,  39, 
183 

Blood,  3,  101-126 ;  coagulation  of,  102, 
104,  194 ;  corpuscles,  103  ;  detection 
of,  120,  121;  gases  of,  127;  specific 
gravity  of,  222  ;  tests  for,  120,  121  ; 
pigment.  111 ;  biological  test  for, 
121  ;  plasma  and  serum  of,  101,  108; 
in  urine,  169;  platelets,  103,  104, 
108,  196 

Blood  bulb,  234 

Blood  clot,  103 

Bohr  on  absorption  of  oxygen,  130  ; 
on  tension  of  carbonic  dioxide,  130 

Bone,  composition  of,  43 ;  marrow,  23 

Boron,  8 

Bowman's  capsule,  143 

Boyle-Mariotte's  law  for  gases,  240 

Bran,  58 

Bread,  50,  52,  58;  composition  of,  59 

Bright 's  disease,  168 

Bromine,  8 

Brown  and  Morris  on  digestion  of  starch, 
68 

Buchner  on  ferments,  66 

Buffy  coat,  103 

Bunge  on  hsematogens,  46 ;  on  milk,  53 

Bush  tea,  60 

Butter,  49,  52 

Butyric  acid,  19,  24,  85 

Butyrin,  55 


Cadmium,  8 

Caffeine,  60,  159 

Calcium,  5,  8 ;  phosphate,  49 


INDEX 


247 


Calcium  caseinogenate,  55,  181 
Calcium  oxalate  in  urine,  156, 162,  165  ; 

crystals,  164 
Calcium  salts,  3, 105  ;  importance  of,  in 

coagulation  of  blood,  104,  105,  194  ; 

of  milk,  55,  181 
Cane  sugar,  13,  16,  18,  74,  96,  171,  177, 

178  ;  urine,  18  ;  tests  for,  13,  18 
Caproic  acid,  24,  32 
Caproin,  55 
Carbamate,  150 

Carbamide,  151.     See  also  Urea 
Carbohydrates,  4,  13-21,  176-179;  ab- 
sorption of,  96  ;  classification  of,  16  ; 

definition  of,  14  ;  tests  for,  13-21,  171, 

176,  177,  178,  179 
Carbolic    acid     poisoning    and     urine, 

216 
Carbon,  3,  8  ;  tests  for,  3,  9 
Carbonates  in  blood,  129, 130  ;  in  urine, 

154 
Carbonic  acid  in  air,  126  ;  in  blood,  129, 

130 
Carbonic  oxide  hssmoglobin,    114,  120, 

172,  188 
Cardiac  glands,  70  ;  muscle,  237 
Carmine-stained  fibrin,  184 
Cartilage,  44 
Casein,  49,  54,  60,  172 
Caseinogen,  10,  35,  49,  64,  65,  172,  181, 

186 
Caseinogenate  of  calcium,  of  sodium,  of 

potassium,  55 
Catalysing  agents,  67,  82 
Catalysts,  67 

Cell,  definition  of,  2  ;  diagram  of,  45 
Cells,  differentiation  of,  1 
Cellulose,  16,  21,  85,  95 
Centigrade  scale,  7 
Central  cells  of  fundus  glands,  70,  71, 

72 
Centrifugal  machine,  195  - 
Centrosome,  2 
Cerebrin,  201 
Cerebrosides,  20] 
Cerebrospinal  fluid,  203 ;  functions   of, 

203 
Chemical  physiology,  1 
Chemical  sediments  of  urine,  165 
Chemical  structure  of  protoplasm,  3 
Chemistry  of  respiration,  126;  of  nerve 

degeneration,  201 
Chittenden's  views  on  diet,  52,  148 
Chlorides  of  urine,  141, 153  ;  estimation 

of,  205  ;  tests  for,  141 
Chlorine,  3,  8 
Chlorophyll,  95 
Cholalic  acid,  91,  94 
Cholesterin,  3,  26,  56,  79,  90,  91,  93,  94, 
95,  111,    201  ;    crystals  of,    93,    173 ; 
tests  for,  93 


Choletelin,  92 

ChoHne,  26,  85,  201  ;  tests  for,  201,  202 
Chondrin,  44 
Chorda  tympani,  68 
Chromatin,  45 
Chromogens  in  urine,  216 
Chromo-proteins,  41,  44 
Chyle,  99 
Chyme,  89,  94 
Cilia,  237 

Ciliary  movement,  237 
Circular  polarisation  and  chemical  con- 
stitution, 230 
'  Circulating  protein,'  148 
Circulation  of  bile,  90 
Cirrhosis  of  liver,  147 
Citric  acid,  54,  144 
Clark's  essence  of  rennet,  60 
Cleavage,  30 ;  products  of  digestion,  81, 

97,98 
Clotting  of  blood,  102-108,  194-196 
Clupeine,  42 
Coagulated  proteins,  43 
Coagulation,  40 
Coagulation  of  blood,  102-108, 194-196 ; 

of  milk,  49,  54,  181 ;  of  muscle,  197- 

198  ;  of  proteins,  27 
Coagulative  ferments,  65 
Cobalt   sulphate,   actions    on   proteins, 

183 
Cocaine,  60 
Cocoa,  60,  159 
Coffee,  60,  159 
Coffin-lid  crystals,  155,  157 
Cola  nut,  60 
Collagen,  43,  81 
Collimator,  116 
Colloid   carbohydrates,   salting   out  of, 

21 
Colloidal  solution,  36 ;  substances,  237, 

238 
Colloids,  37,  38,  243 
Colostrum,  4,  53 
Colostrum  corpuscles,  53 
Commercial  peptone,  37,  107 
Complement,  124 
Compounds  found  in  the  body,  4 
Condiments,  60 
Conjugated  proteins,  41,  44 
Cooking  of  food,  59 
Copper,  3,  8 
Cream,  49,  53 
Creatine,    32,    60,    110,    152,   200;     in 

muscle,  200 
Creatinine,  30,  110,  142,  145,  148,  149, 

152,  168,  174,  210 ;  detection  of,  142  ; 

estimation  of,  211 
Cresol,  66 

Croft  Hill  on  ferments,  66 
Crypts  of  Lieberkiihn,  84 
Crystallin,  43 


248 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


Crystalline  lens,  43 

Crystallisable  proteins,  38, 112, 113,  187, 

191 
Crystallisation  of  egg  albumin,  38,  180 
Crystalloids,  38,  238 
Crystals  from  blood,  112,  113,  187,  191 
Curative  inoculation,  121 
Curd,  54,  181 
Curds  and  whey,  54 
Cyclopterine,  42 
Cystin,  34,  35,  162,  163,  165  ;    crystals 

of,  164 
Cystinuria,  164 
Cytosine,  34,  46 


Decalcified  blood,  101,  104,  194 

Decalcified  milk,  49,  181 

Decomposition  products  of  fats,  25 

Density  of  water,  8 

Dentine,  composition  of,  43 

Deposits  in  urine,  162-165 

Desiccator,  199 

Deutero-albumose,  75,  77 

Deutero-proteose,  81,  168,  172,  187 

Dextrin,  13,  16,  21,  68,  171,  178;  tests 
for,  21 

Dextro-rotatory,  17  ;  quartz,  226 

Dextrose,  13,  16,  17,  88,  110,  168,  171, 
177,178;  crystals,  17;  in  blood,  17, 
110 ;  in  urine,  166,  168 

Diabetes  mellitus,  17,  87 

Diabetic  coma,  88 

Diabetic  urine,  88,  166,  168 

Diacetin,  25 

Dialyser,  37 

Dialysis,  37,  38,  192,  236,  238,  239; 
of  proteoses,  182 ;  of  serum,  192 

Di-amino  acids,  32,  34 

Di-amino-caproic  acid,  32 

Di-amino-valeric  acid,  32,  150 

Diastase,  65 

Diastatic  ferments,  19,  68,  179 

Diet,  51 

Diet  tables,  51 

Dietary,  51 

Diffusion,  130,  236,  238 

Digestion,  184;  gastric,  62,  70-77;  in- 
testinal, 84-86  ;  pancreatic,  78-84  ; 
salivary,  62-70 

Diraethylxanthine,  159 

Dioxy-purine,  159 

Dipeptide,  36 

Diphtheria-toxin,  122 

Direct-vision  spectroscope,  117 

Disaccharides,  16,  18,  66 

Dissociation,  236,  237  ;  of  oxygen,  133 

Disuse  atrophy,  203 


Divalent  elements,  236 
Dough,  58 
Dripping,  59 
Dropsical  effusions,  45 
Dulcite,  15 

Dumb-bell  crystals,  157 
Dupr6's  urea  apparatus,  141 
Dysalbumose,  182 


E 


149 


Eck's  fistula, 

Edestin,  35 

Egg-albumin,  35,  43,  96  ;  crystallisation 
of,  180 

Egg-globulin,  27,  28 

Eggs,  50,  56 

Egg-white,  27,  28,  62,  78 

Egg  yolk,  44 

Ehrlich's  experiments  with  methylene 
blue,  128  ;  hypothesis,  123 

Elastic  fibres,  44 

Elastin,  44,  75,  81,  95 

Elastoses,  75 

Electrolytes,  237 

Electrolytic  action,  238 

Elements  found  in  the  body,  3 ;  sym- 
bols and  atomic  weights  of,  8 

Emulsification,  23,  25,  99 

Emulsion,  82 

Enamel,  composition  of,  43 

Endogenous  metabolism,  149 ;  uric 
acid,  160 

English  system  of  weights  and  measures, 
6,7 

Entero-kinase,  84,  105,  186 

Envelope  crystals,  156 

Enzymes,  63,  66,  67,  68 

Epidermis,  2 

Epithelium  of  intestine,  98,  99 

Epsom  salts,  153 

Erepsin,  97,  161 

Erlenraeyer  flask,  2]  1 

Erythro-dextrin,  21,  68,  179 

Esbach's  albuminometer,  5,  166 :  re- 
agent, 5  ;  tube,  166 

Estimation  of  chlorides,  205  ;  of  crea- 
tinine, 211 ;  of  dextrose,  166,  167  ;  of 
lactose,  19,  179  ;  of  maltose,  20, 179  ; 
of  nitrogen,  235  ;  of  phosphates,  207  ; 
of  sulphates,  208  ;  of  urea,  204  ;  of  uric 
acid,  210 

Ethereal  sulphates  in  urine,  153,  154 ; 
estimation  of,  208 

Ethyl  alcohol,  24 

Ethyl-diacetic  acid,  88,  168 

Eu-globulin,  193 

Exogenous  katabohsm,  148 

Exogenous  uric  acid,  160 


INDEX 


249 


Expired  air,  126 
External  respiration,  126 
Extirpation  of  pancreas,  87 
Extractives   of   blood,   110;   of  muscle, 

60 
Extraordinary  ray,  222 


F^CES,  94 
Fahrenheit  scale,  7 
Fatigue,  201 

Fat,  2,  96  ;  absorption  of,  99  ;  constitu- 
tion, 24  ;  decomposition  products  of, 

25  ;  melting  point  of,  23 ;  tests  for, 

22  ;  digestion  of,  100 
Fat-splitting  ferment,  80 
Fats,  4,  22-26,  78,  93  ;  of  milk,  55 
Fatty  acids,  22,  94,  100 
Fehling's  test,  33, 17, 19,  168  ;  solution, 

166 
Ferment  coagulation,  40 
Ferment,  invert,  84 
Ferment  of  ferments,  84 
Fermentation,  63 
Fermentation  test,  13,  169 
Ferments,  63-67  ;  of  gastric  juice,  65  ; 

of  pancreatic  juice,  65  ;  of  saliva,  65 ; 

of   succus   entericus,   65 ;   organised, 

64 ;  unorganised,  65 
Ferments,    classification   of,  64 ;    dias- 

tatic,  19,  68,  179 
Fertilisation,  237 
Fibres,  elastic,  44 
Fibrin,  78,  101,  103,  106,  108,  184 
Fibrin  ferment,  40,  65,  101,  103,  104, 

105,  106,  108,  109,  110 ;  preparation 

of,  101 
Fibrin  filaments,  103 
Fibrinogen,  43,  104,  106,  108,  109,  172 
Fibrino-globulin,  104 
Filtration,  23B,  237 
Fischer  on  proteins,  31 
Fistula,  gastric,  70 
Fleischl's    haBmometer,    220 ;    spectro- 

polariraeter,  229 
Flour,  50,  57,  58 
Fluorescent  screens,  191 
Fluorine,  3 

Folin's  method  of  estimating  urea,  204 
Foods,  49 ;  cooking  of,  59  ;  accessories 

of,  60 
Formaldehyde,  4  ;  reaction,  27,  39 
Formic  acid,  24 
Fractional  heat  coagulation  of  muscle 

proteins,  198  ;  of  serum  proteins,  192, 

193 
Fraunhofer's  lines,  102,  115,  119 
Fredericq  on  tension  of  carbon  dioxide, 

130 


Free  hydrochloric  acid  in  gastric  juice, 

tests  for,  185 
Fundus  glands,  70,  71,  72 
Fiirth  on  muscle  plasma,  195 


Galactose,  15,  16,  18,  178,  201 
Gallstones,  93 

Gamgee  on  photographic  spectra,  190 
Garrod  and  Hopkins  on  urobilin  and 

stercobihn,  213,  214 
Garrod  on  urochrome,  213 
Garrod's  method  of  separating  pigments 

from  urine,  213 
Gas,  analysis  of,  234 
Gaseous  interchange  in  lungs,  128,  129, 

130 
Gases  in  blood,  109,  127 
Gastric  digestion,  62 
Gastric  fistula,  70 
Gastric  juice,  70,  85,  185  ;  action  of,  75; 

composition  of,  73  ;  glands,  70,  71  ; 

properties  of,  74  ;  secretion  of,  70 
Gastrin,  84 

Gay-Lussac's  law,  240 
Gelatin,  30,  35,  48,  44,  75,  173;  tests 

for,  30 
Gelatinisation,  30 
Gelatoses,  75 

Germ  theory  of  disease,  63 
Gliadin,  35,  48,  58 
Globin,  42,  113 
Globulicidal  power,  122 
Globulin,  37,  41, 42,  75,  77, 123, 171,  200 
Globulose,  75 

Glossopharyngeal  nerve,  67 
Gluco-proteins,  41,  45 
Glucosamine,  45 
Gluccse,  16,  65,  96,  177,  178 
Glutamic-acid,  32,  35,  81 
Gluten,  48,  50,  58 
Gluten- fibrin,  58 
Glyceric  ethers,  24 
Glycerides,  24 

Glycerin,  25,  85,  100  ;  tests  for,  22 
Glycerol,  25 

Glycine,  31,  35,  41,  91,  94,  150 
Glycocholate  of  soda,  90,  91 
Glycocholic  acid,  91 ;  preparation  of,  79 
Glycocine,  see  Glycine 
Glycogen,  14,  21,  65,  96,  161,  171,  176, 

177  ;  microscopical  detection  of,  177  ; 

preparation  of,  176 ;  tests  for,  14,  21 
Glycol,  15 
Glycolysis,  88 
Glycosuria,  88 

Glycuronic  acid,  4,  87,  88,  169,  178 
Glycyl,  36 


250 


ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 


Glyoxylic  acid,  4.  27 

Gmelin's  test,  79,  92,  169 

Gnezda  on  biuret  reaction,  183 

Goblet  cells,  45 

Gold,  8 

Gowers,  Sir  William,  his  hgemacyto- 
meter,  217  ;  hsemoglobinometer,  219 

Graham,  Thomas,  on  colloids  and  crys- 
talloids, 38 

Gram-molecular  solutions,  238 

Granulose,  20 

Grape-sugar,  see  Dextrose 

Green  vegetables,  61 

Ground  substance,  30 

Griitzner's  method  of  comparing  diges- 
tive power  of  solutions  of  pepsin,  184 

Guanase,  161 

Guanine,  47,  159,  160,  161 

Guarana,  60 

Gums,  95 

Gunsberg's  reagent,  185 

Giirber  on  serum  albumin  crystals,  39 


H 


Ha:macytometer  of  Sir  William  Gowers, 

217  ;  of  Oliver,  218 
Heematin,   95,    113,    213;    acid,    188; 

alkaline,  188  ;  iron  free,  191 ;  of  food, 

143 
Haematogen,  46 
Haematoidin,  114  ;  crystals,  89 
Haematoporphyrin,  113,  191,  214,  215, 

216 ;  absorption  bands  of,  189,  215 
Haematoscope  of  Hermann,  117 
Haemin,   101,  113 ;   crystals,  113 ;  pre- 
parations of.  102,  113 
Haemochromogen,  190,  200 
Haemoglobin,  11,  38,  92,  95,  102,  112, 

114,  188 ;  composition  of,  113  ;  deri- 
vatives of,  188 
Haemoglobinometer     of     Sir     William 

Gowers,  219 ;  Haldane,  220  ;  of  Oliver, 

220 
Haemoglobinuria,  122,  170 
Haemopyrrol,  93,  113,  114 
Hasmolysins,  122 
Haemometer  of  von  Fleischl,  220 
Haldane  on  metheemoglobin,  120 ;    on 

absorption  of  oxygen,  130  ;  on  oxygen 

tension,  134 
Haldane's  haemoglobinometer,  220 
Hamburger  on  digestive  action  of  juices, 

184 
Hammarsten's  method  of  precipitating 

serum  globulin,  192 
Hammerschlag's  method  of  estimating 

the  specific  gravity  of  blood,  222 
Haptophor  group,  123 
Hayem's  fluid,  219 


Hay's  test  for  bile  salts,  79,  169 

Heart  muscle,  199 

Heat  coagulation  of  proteins,  37,  192, 

193,    198  ;    of    serum    globulin   and 

serum  albumin,  192,  198 
Heat  rigor,  198 
Heidenhain  on  secretion  of  gastric  juice, 

72  ;  on  pressure  in  bile  duct,  89 
Heller's  nitric  acid  test,  166 
Henry-Dalton  Law,  240 
Heptoses,  16 

Hermann,  haematoscope  of,  117 
Hetero-albumose,  75,  76,  182  ;  proteose, 

81 
Hexone  bases,  33,  84,  150 
Hexoses,  16 
Hill's  air-pump,  233 
Hill,  Croft,  on  ferments,  66 
Hippuric  acid,  162,  173 ;  tests  for,  162 
Hirudin,  107,  195 
Histidine,  32,  33,  41 
Histone  of  Kossel,  41,  42,  113 
Hofmeister    on    crystallisation   of    egg 

albumin,  38 
Homogentisinic  acid,  169 
Hopkins's   method   of    estimating  uric 

acid,     158,      210 ;    on    Adamkiewicz 

reaction,  27 ;  on  crystallisation  of  egg 

albumin,  39,  180 ;  reaction  for  lactic 

acid,  185 
Hoppe-Seyler  on  proteins,  30 
Hot-air  oven,  176 
Howell  on  rhythmical  action,  237 
Htifner's    method    of  estimating  urea, 

141 
Hyaline  cartilage,  44 
Hydrazone,  177 
Hydrobilirubin,  92,  93,  94,  143 
Hydrocele  fluid,  108 
Hydrochinon,  216 
Hydrochloric  acid,  70 

of  gastric  juice,  72,  73 

tests  for  cane  sugar,  13 
Hydrogen,  3,  8  ;  tests  for, 
Hydrolysis,  19,  75 
Hydrolytic  ferments,  65 
Hydrometer,  49,  53,  141 
Hydroxybutyric  acid,  88, 
Hydroxyl,  24 
Hypertonic  solutions,  242 
Hypobromite   of    soda,    action    of,    on 

urea,  141,  146 
Hypotonic  solutions,  242 
Hypoxanthine,  47,  60,  159,  160,  161 


Immune  body,  124 

Immunity,  121,  123  ;  side  chain,  theory 
of,  123 


formation  of,  71 
tests  for,  185 


9 


168 


INDEX 


251 


Indican,  154,  216 

Indiffusibility  of  proteins,  38,  244 

Indigo,  154  ;  blue,  216  ;  red,  216 

Indole,  85,  95,  154 

Indole-amino-propionic  acid,  39 

Indoxyl,  154,  216 

Indoxyl  sulphate  of  potassium,  154,  216 

Infection,  122,  123 

Infra-proteins,  47,  48,  171 

Inoculation,    curative,   and    protective, 

121-126 
Inorganic  compounds,  4 ;  salts,  4 
Inosite,  14.  18 ;  crystals,  18 
Inspired  air,  126 
Internal  respn-ation,  126 
Internal  secretion  of  pancreas,  88 
Interstitial  substance,  30,  45 
Intestinal  juice,  18,  84  ;  digestion,  84 
Intravascular  coagulation,  103,  105,  196 
Inversion,  17,  18,  84 
Inversion  of  cane  sugar,  18,  65 
Inversive  ferments,  65,  66 
Invertin,  65,  84 
Involuntary  muscle,  198 
Iodine,  3,  8,  11  . 
Iodine  test,  13,  14,  176,  179 
Ionic  action,  237 
lonisation,  73 
Ions,  73,  236,  240 
Iron,  3,  8,  112,  113  ;  in  milk,  53 
Iron-free  hsematin,  191 
Islets  of  Langerhans,  79,  87 
Iso-cholesterin,  93 
Isotonic  solutions,  241 
Isotropous  bodies,  223 


Jaffe  on  test  for  indoxyl,  216 

Jaundice,  169 

Jelly,  Whartonian,  30 

Johnson,  G.  S.,  on  creatinine,    152  ;  on 

sugar  in  urine,  167 
Juice,  intestinal,  18,  84 
Junkets,  60 


Karyokinesis,  237 

Katabolites,  30,  148 

Rations,  236 

Kauder's  method  of  precipitating  serum 

globulin,  192 
Kephalin,  201 
Keratin,  2,  11,  35,  44,  95 
Ketone,  14 
Ketoses,  15 
Kidney,  142 
Kidneys  removal  of  part  of,  87  ;  removal 

of  both,  147 


Kjeldahl's  method  of  estimating  nitro- 
gen, 235 

Kossel  on  protamines,  41  ;  on  histone, 
113 

Koumiss,  56,  60 

Kiihue  on  precipitation  of  pepsin,  74 

Kiilz's  method  of  extracting  glycogen, 
176 


Lactalbumin,  42,  64, 181  ;  properties  of, 

54 
Lacteals,  95,  99 
Lactic  acid,  4,  53,  56,  60,  95,  158,  185 ; 

Hopkins's  reaction  for,  185, 197 ;  tests 

for,  185 
Lactic  acid  fermentation,  19  ;  in  milk, 

56  ;  in  muscle,  197  ;  organisms,  19 
Lacto-globulin,  181 
Lactometer,  49 
Lactose,  13,  16,  19,  49,  56,  65,  171,  177, 

178;    in   urine,    168;    tests  for,  19, 

177 
Laky  blood,  112 
Lanoline,  93 
Lard,  22 

Lateritious  deposit,  156 
Laurent's  polarimeter,  228 
Lead,  3,  8 
Lecithin,  3,  4,  10,  25,  26,  56,  85,  91,  93, 

111,  155,  201 
Leech  extract,  107,  194 
Lethal  dose,  122 
Leucine,  31,  80,  81,  84,  85,  86,  148,  165, 

187 ;  as  a  urea  precursor,  150  ;  tests 

for,  187 
Leucine  crystals,  86,  150  ;  preparation 

of,  187 
Leucocytes,  160,  161 
Leucocythoemia,  160 
Leucyl,  36 

Levo-rotatory,  17  ;  quartz,  226 
Levulose,  16,  17,  171, 177  ;  in  blood,  17 ; 

in  muscle,  17  ;  in  urine,  17 ;  reactions 

of,  17 
Lieberkiihn's  crypts,  84  ;  jelly,  48 
Liebermann's  reaction,  79 
Liebig's  extract,  200 
Lime  water,  30 
Lipochrome,   55,   56,   200 ;   in   muscle, 

200 
Lipolytic  ferments,  65 
Liquor  pancreaticus,  78,  187  ;  pepticus, 

62 
Lithates,  see  Urates,  156 
Lithium,  3,  8 

Litre,  standard  of  capacity,  7 
Liver,  function  of,  in  relation  to  urea, 

147  ;  uric  acid,  158 
Living  test-tube  experiment,  109 


252 


ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 


Loeb  on  ionic  action,  237  ;  on  fertilisa- 
tion, 237 
Loewy's  aerotonometer,  132 
Lungs,  126 

Lymph  formation,  242 
Lysine,  32,  33,  81 


M 


McMuNN  on  stercobilin,  95 

McWilliam's  test  for  proteins,  183 

Magnesium,  3,  8 

Magnesium  phosphate,  165 

Magnesium  sulphate,  action  of,  on  pro- 
teins, 27,  40,  49 

Malic  acid,  231 

Mallow,  18 

Malpighian  capillaries,  142 

Malt  upon  starch,  action  of,  179  ;  dias- 
tase, 179 

Maltase,  65 

Malting  ferment,  179 

Maltose,  13,  16,  19,  65,  66,  68,  78,  171, 
177,  178,  179 

Maly  on  the  formation  of  hydrochloric 
acid,  72,  73 

Mammary  glands,  84,  88 

Manganese,  3,  8 

Mannite,  15 

Mannose,  15 

Marchi  reaction,  202 

Marsh  gas,  65 

'  Mass  Action,'  73 

Measures  of  capacity,  7  ;  of  length,  7 

Meat,  50,  52,  67  ;  constituents  of,  57 

Meconium,  95 

Melanin,  216 

Mercurial  air-pumps,  231 

Mercury,  8 

Metabolism,  2 

Meta-proteins,  see  Infra-proteins 

Methffimoglobin,  114,  119,  170,  172, 
191 ;  in  urine,  170 

Methane,  65,  85 

Methyl-indole,  34 

Methyl-glycine,  32 

Methylene  blue  experiments,  128 

Methyl-guanidine  acetic  acid,  32 

Methyl-orange,  235 

Metric  system,  7 

Mett's  tubes,  184 

Microchemical  detection  of  glycogen, 
177 

Micrococcus  urea,  66 

Microspectroscope,  117 

Milk,  23,  49,  50,  52,  53,  181 ;  alcoholic 
fermentation  of,  56 ;  coagulation  of, 
54,  181  ;  composition  of,  54 ;  fats  of. 


54 ;    proteins   of,  54 ;    salts  of,   56 ; 

souring  of,  56  ;  sugar,  56 
Milk-curdling,  ferment  of  pancreas,  80, 

82  ;  of  stomach,  49,  54,  55 
Milk-sugar,  19,  49,  56  ;  crystals,  19,  96 
Millon's  reagent,  5,  27  ;    test,  27 ;    re- 
action, 39 
Mineral  compounds,  3 
Mohr's  method  of  estimating  chlorides, 

205 
Monatomic  alcohols,  24 
Monoacetin,  25 
Monoamino-acids,  32,  34 
Monoamino-caproic  acid,  32 
Monochromatic  light,  228 
Monosaccharides,  16,  17,  66 
Monovalent,  236 
Monoxypurine,  159 
Moore  and  Rockwood  on  fat  absorption, 

100 
Moore  on  osmotic  pressure  of  proteins, 

243 
Moore's  test  for  sugar,  13,  17 
Morner  and  Sjoqvist's  method  of  esti- 
mating urea,  204 
Morris  and  Brown  on  starch  digestion, 

68 
Mucedin,  58 
Mucic  acid,  18,  178 
Mucin,  2,  30,  45,  67,  68,  91,  94,    95, 

173  ;  in  bile,  91  ;  in  saliva,  62,  67  ; 

in  urine,  162  ;  tests  for,  62 
Mucinogen,  67 
Mucoids,  45 

Mucous  glands,  structm-e  of,  45,  69 
Mucous  membrane  of  frog's  intestine, 

99 
Mucous  salivary  glands,  69 
Mucus,  170 

Munk  on  fat  absorption,  100 
Murex,  157 
Murexide  test,  156 
Muscle,    199 ;   clot,    198 ;  pigments   of, 

199 ;  plasma  of,  197 ;  extractives  of, 

200 
Muscle,  cardiac,  237 
Muscle  fibrin,  198 
Muscle  sugar,  18 
Muscle  tissue,  197 
Muscles,  pale  and  red,  199  ;  involuntary, 

198 
Muscular  movement,   2 ;   exercise  and 

carbonic  acid,  136 
Mutton,  57 

Myogen,  198  ;  myogen  fibrin,  198 
Myo-haematin,     199,    200 ;    absorption 

spectrum  of,  200 
Myosin,   4,   57,   75,   197,    198;    fibrin, 

198 
Myosinogen,  37,  198 
Myxoedema,  87 


INDEX 


253 


N 


Negative  effect,  104 

Nencki  and  Sieber  on  pigments,  113 

Nencki's  experiment  on  urea,  150 

Nerve  degeneration,  chemistry  of,  201 

Nervous  impulse,  238 

Nervous  tissue,  197,  200;  composition 
of,  200 

Neurokeratin,  44 

Neutral  fats,  23 

Neutral  salts,  action  of,  on  carbo- 
hydrates, 21 ;  on  proteins,  27,  28,  37, 
43,  49,  77,  101,  102,  182,  192,  193 

Nickel,  8 

Nickel  sulphate,  action  of,  on  proteins, 
183 

Nicol's  prism,  223 

Nissl's  bodies,  201 

Nitrate  of  urea,  146,  156 

Nitric  oxide  haemoglobin,  114,  120 

Nitrogen,  3,  8 ;  estimation  of,  235 ; 
tests  for,  9,  10 

Nitrogenous  food,  50,  51 ;  glucosides, 
201 

Nitrous  acid,  action  of,  on  urea,  146 

Non-electrolytes,  237 

Nuclease,  161 

Nucleic  acid,  45,  46  ;  decomposition  of, 
47 

Nuclein,  3,  45,  155,  160, 161 

Nucleo-protein,  3,  10,  41,  45,  46,  91, 
103,  105,  109,  173,  196,  200;  in 
bile,  79,  91 ;  decomposition  of,  47  ;  in 
muscle,  198  ;    tests  for,  79,  173 

Nucleus,  functions  of,  2 


0 


Oatmeal,  52 

Oleic  acid,  24 

Olein,  23,  55,  203 

Olfactory  nerve,  67 

Oliver's  hamacytometer,  218 ;  ha?mo- 
globinometer,  220 

Opsonins,  125 

Orcin  reaction,  178 

Ordinary  ray,  222 

Organic  compounds,  4 

Organised  ferments,  64 

Ornithine,  32,  150,  161 

Osazones,  177 

Osborne,  W.  A.,  on  coagulation  of  milk, 
55 

Osmic  acid  test,  22,  24 

Osmium,  8 

Osmosis,  38,  236,  239 

Osmotic  pressure,  238,  239,  243  ;  cal- 
culation of,  240  ;  of  proteins,  243  ; 
determination  of,  by  freezing  point, 
241 ;  physiological  application,  242 


Ossein,  43 

Osteomalacia,  168 

Ovarian  cyst  fluid,  45 

Ovo-mucoid,  56 

Oxalate  of  calcium,  in  milk,  47,  55; 
plasma,  106  ;  in  urine,  156,  164 

Oxalate  of  urea,  146,  156 

Oxidases,  66,  161 

Oxygen,  3,  8 ;  in  blood,  128 ;  tension, 
137 

Oxyhsemoglobin,  101,  102,  112,  113, 
114,  169,  170,  172,  188,  191 ;  crystals, 
102,  112,  187;  in  muscle,  200;  pre- 
paration of,  191 

Oxyhtemoglobin  pure,  preparation  of, 
191 

Oxyntic  cells  of  fundus  glands,  70,  72, 
73 

Oxyphenyl-alanine,  34 


Pale  muscle,  199 

Palmitic  acid,  24 

Palmitin,  23,  55 

Pancreas,  extirpation  of,  87  ;  grafting 
the,  87 ;  structure  of,  79,  80 

Pancreatic  digestion,  78,  186,  187 ;  se- 
cretion, 186 

Pancreatic  juice,  81,  85  ;  action  of,  78, 
81 ;  composition  of,  80  ;  secretion  of, 

.  83 

Panum's  method  of  precipitating 
serum  globulin,  192 

Paraglobulin,  see  Serum  globulin 

Para-mucin,  45 

Paramyosinogen,  43,  197,  198 

Parietal  cells  of  fundus  glands,  70,  71 

Parotid  gland,  67 

Partial  pressure  of  gases,  126-140 

Pasteur  on  circular  polarisation,  230 

Pathological  urine,  166  ;  pigments  of, 
216 ;  pigments,  216 

Pavy  on  glycogenic  function  of  liver  96, 
176  ;  an  estimation  of  sugar,  167 

Pavy's  solution,  167 

Pawlow  on  secretory  nerve-fibres  to  the 
gastric  glands,  74  ;  to  the  pancreas,  83 

Peas,  58 

Pentoses,  16,  178 

Pepsin,  65,  70,  72,  74,  81  ;  solutions, 
activity  of,  184 ;  hydrochloric  acid, 
70,  74,  82 

Pepsinogen,  72 

Peptic  digestion,  75,  185 

Peptides,  36 

Peptone,  28,  31,  37,  47,  65,  75,  76,  77, 
85,  96,  107,  171,  187,  195  ;  precipita- 
tion of,  76  ;  in  urine,  168  ;  tests  lor,  27, 
76 


254 


ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY 


Peptone  blood,  195 

Peptonuria,  168 

Perfect  foods,  50,  53 

Pericardial  fluids,  108 

Pettenkofer's  test  for  bile  salts,  79,  91, 
92,  169 

Pfliiger's  mercurial  air-pump,  231,  232 

Phagocytes,  121,  122 

Phenol,  66,  85,  95 

Phenolphthalein,  22  ;  phenyl-alanine, 
33,  34,  98 

Phenyl  sulphate  of  potassium,  154 

Phenyl-hydrazine  test  for  sugar,  177 

Phloretin,  88 

Phloridzin,  88  ;  diabetes,  88 

Phloroglucin  reaction,  178 

Phosphates  of  urine,  141,  154, 156,  163  ; 
estimation  of,  207  ;  tests  for,  141,  156, 
163,  165 

Phosphates,  stellar,  155,  156,  164,  165 

Phosphates,  triple,  155,  156,  164,  165 

Phospho-molybdic  acid,  182 

Phospho-proteins,  41,  44 

Phosphorised  fat,  201 

Phosphorus,  3,  8,  11  ;  tests  for,  10 

Phospho-tungstic  acid,  182 

Photographic  spectra,  191 

Physiological  chemistry,  1 

Physiological  proximate  principles,  de- 
tection of,  171 

Pickering  on  colour  reactions  of  proteins, 
183 

Picramic  acid,  17 

Picric  acid,  17  ;  tests  for  sugar,  17,  167 

Pigment  of  red  corpuscles,  110-114  ;  of 
muscle,  199  ;  of  urine,  213,  215 

Pigments,  pathological,  215 

Pilocarpine,  administration  of,  80,  187 

Piotrowski's  reaction,  27,  41,  183 

Plain  muscle,  198,  199 

Plasma,  constituents  of,  109  ;  gases  of, 
109  ;  proteins  of,  109 

Plasma  of  blood,  101,  103,  108,  109, 
194  ;  of  muscle,  197 

Plasmon,  186 

Platinum,  8 

Pleochromatism,  225 

Poisonous  alkaloids,  85 

Polarimeters,  178, 226  ;  of  Laurent,  228  ; 
of  Soleil,  227  ;  of  Zeiss,  228 

Polarisation,  circular,  and  chemical  con- 
stitution, 230 

Polarisation  of  light,  222 

Polariscopes,  226 

Polarised  light,  222  ;  action  of  carbo- 
hydrates on,  16,  17,  18,  19  ;  of  pro- 
teins, 39 

Polarising  microscope,  224 

Polypeptides,  31,  36,  47 

Polysaccharides,  16,  20 

Pork,  57 


Portal  vein,  89 

Positive  phase,  104 

Potash,  method  of  extracting  glycogen, 
176  ;  of  showing  zymogen  granules, 
187 

Potassio-mercuric  iodide,  182 

Potassium,  3,  8 

Patassium  caseinogenate,  55 

Potassium  ferricyanide  in  preparation 
of  methaemoglobin,  188 

Potassium  ferrocyanide  in  the  estimation 
of  phosphate,  207 

Potassium  permanganate  in  estimation 
of  uric  acid,  210,  211 

Potassium  sulphocyanide  in  saliva,  62, 
68 

Potatoes,  52 

Precipitants  of  proteins,  39,  40,  182, 
183 

Precipitation,  40 

Precipitin,  125 

Prevost  and  Dumas  on  formation  of 
urea,  147 

Primary  albumoses,  75;  proteoses,  75 

Principal  cells  of  stomach,  70,  71 

Prisms  in  direct-vision  spectroscope, 
117 

Propionic  acid,  24,  31,  33 

Prosecretin,  83,  186 

Prosthetic  group,  44 

Protagon,  201 

Protamines,  41,  42 

Protective  inoculation,  121 

Protein-hydrolysis,  47 

Protein  metabolism,  3 

Proteins,  3,  9,  11,  27-48,  57,  68-,  81,  85, 
93,  96,  98,  123,  171,  238,  244;  absorp- 
tion of,  96,  97  ;  classification  of,  41 ; 
coagulated,  37  ;  coagulation  of,  37 ; 
colour  reactions  of,  27,  39  ;  composi- 
tion of,  3,  30 ;  conjugated,  44  ;  crys- 
tallisation of,  38  ;  definition  of,  30 ; 
digestion  of,  by  gastric  juice,  75 ;  diges- 
tion of,  by  pancreatic  juice,  81 ;  heat 
coagulation  of,  37  ;  of  blood  plasma, 
109;  of  milk,  54  ;  of  muscle,  197,  198  ; 
of  serum,  109  ;  osmotic  pressure  of, 
243  ;  in  urine,  168 ;  precipitants  of, 
27,  39,  40  ;  sclero-,  43  ;  simple  solu- 
bilities of,  36 ;  tests  for,  14,  36,  182, 
183 

Protein-sparing  food,  44 

Proteolytic  ferments,  74j  enzymes,  74, 
81,  161 

Proteoses,  31, 37, 47,  65, 75,  76, 77,  79, 97, 
107,  171,  172,  182,  195 ;  in  urine,  168 

Prothrombin,  105 

Proto-albumoses,  75,  76,  182 

Protones,  41 

Protoplasm,  chemical  structure  of,  3  ; 
properties  of,  2 


INDEX 


255 


Proximate  principles,  classification  of,  4  ; 

of   food,   50 ;    scheme   for  detecting, 

171-174 
Pseudo-globulin,  193,  198 
Pseudo-mucin,  45 
Ptomaines,  63 
Ptyalin,  19,  05,  68,  69 
Pulses,  58 

Pumps,  mercurial  air-,  231-234 
Purine  bases,  44,  46,  47,  159,  161 
Purpurate  of  ammonia,  156 
Pus  in  urine,  170;  tests  for,  170 
Putrefaction,  19,  66,  85 
Pyloric  glands,  70,  71 
Pyrimidine  bases,  34 
Pyrocatechin,  216 
Pyrrolidine  derivatives,  34 


Q 


QUADRIURATES,  158 

Quantitative  estimation  of  albumin,  166  ; 
of  chlorides,  205  ;  of  creatinine,  211  ; 
of  dextrose,  167 ;  of  glycogen,  176, 
177 ;  of  lactose,  19,  179 ;  of  maltose, 
20,  179;  of  nitrogen,  235;  of  phos- 
phates, 209  ;  of  sulphates,  210  ;  of 
sugar,  167  ;  of  urea,  205  ;  of  uric  acid, 
210 


R 


Kanke's  diet,  51 

Reagents  necessary  for  practical  work, 

4,  5,  6 
Reaumur's  scale,  7 
Receptor  group,  123 
Red  blood  corpuscles,  110  ;  composition 

of,  110  ;  pigment  of,  110 
Red  muscle,  199 
Reflex  action,  67 

Reid,  Wavmouth,  on  absorption,  243 
Rennet,  40,  60,  65,  72 
Rennin,  60,  72,  82 
Reproduction,  2 
Resins,  95 
Respiration,  chemical  stimulus  to,  138  ; 

chemistry  of,  126  ;  in  excised  tissues, 

138 
Respiratory  oxygen  of  haemoglobin,  114 
Respiratory  pigments,  112 
Respiratory  quotient,  127 
Reversible  action,  66 
Rhythmical  contraction,  237 
Ricin,  123 
Rigor  mortis,  198 
Ringed  amino  acids,  34 
Ringer  on  contractile  tissue,  237 
Riva  on  urochroiij^  143 


Roberts,  Sir  William,  on  estimation  of 

sugar  in  urine,  169 
Rock  wood  and  Moore  on  fat  absorption, 

100 
Rose's  test  for  protein,  27,  41,  183 
Rosenheim's  formaldehyde  reaction,  27 


Saccharic  acid,  18 

Saccharimeters,  230 

Saccharoses,  16,  65,  84 

St.  Martin,  Alexis,  case  of,  70 

Salicyl-sulphonic  Acid,  action  of,  on 
proteins,  183 

Saliva,  30,  62,  67 ;  action  of,  68,  70  ; 
composition  of,  67  ;  secretion  of,  67 

Salivary  corpuscles,  67 ;  glands,  2,  67, 
69,  107 

Salkowski's  reaction  for  cholesterin,  79, 
93  ;  method  of  estimating  sulphates, 
209 

Salmine,  42 

Salted  plasma,  101 

Salted  whey,  54 

Salting-out  of  colloid  carbohydrates,  21 
of  proteins,  40,  55,  193 

Salts  of  milk,  56 

Saponification,  22,  82,  99 

Sarcine,  159 

Sarcolemma,  44 

Sarco-lactic  acid,  197 

Sarcosine,  32 

Schafer  on  fat  absorption,  98,  C9 

Schili  on  bile  circulation,  90,  94  test 
for  uric  acid,  156 

Schizomycetes,  forms  of,  64 

Schmalz's  capillary  picnometer,  222 

Schmidt,  Alexander,  on  precipitating 
serum  globulin,  192 ;  on  salts  of 
plasma,  110  ;  on  preparation  of  fibrin- 
ferment,  110 

Schroder's  work  on  urea,  150 

Schiitz's  law,  184 

Schwann,  white  substance  of,  93 

Sclero-proteins,  41,  43 

Sebum,  93 

Secretion,  83,  84,  89,  186 

Secretion,  internal,  87 

Sediments  in  urine,  165 

Semipermeable  membranes,  239 

Serine,  32 

Serous  glands,  69 

Serum,  101,  103,  108,  192;  albumin, 
35,  37,  42,  101,  109,  168  ;  crystallisa- 
tion of,  38 ;  gases  of,  109 ;  heat 
coagulation,  37,  42,  193 

Serum,  bactericidal,  globulicidal,  124 

Serum  casein,  192 


256 


ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 


Serum  globulin,  35,  37,  42,  43,  101,  102, 

109,  168  ;  heat  coagulation,  192,  193 
Serum  lutein,  101 
Serum  of  blood,  101,  102  ;  proteins  of, 

101,  102 
Serum  proteins,  109  ;  separation  of,  101 
Sheep's  wool-fat,  93 
Shell  of  eggs,  56 
Side-chain  theory,  123 
Silicon,  3,  8 
Silver,  8 
Silver    nitrate    method    of    estimating 

chlorides,  205 
Skatole,  34,  85,  95 
Skatoxyl,  216  ;  red,  216 
Skimmed  milk,  49,  53 
Smoky  urine,  169 
Snake  venom,  64,  121,  122 
Soap,  22,  25,  82,  90,  100 
Sodium,  3,  8 

Sodium  bicarbonate  in  blood,  129,  130 
Sodium  chloride,  3  ;  action  on  proteins, 

109,    182 ;     on    nucleo-proteins,    46, 

196 
Sodium  hypobromite,  method  of   esti- 
mating urea,  141, 142 
Sodium  phosphate,  29,  30 ;  acid,  144 
Sodium  sulphate  plasma,  101,  104 
Solar  spectrum,  119,  189 
Soleil's  saccharimeter,  227 
Soluble  starch,  20 
Solutions,  286 
Sorbite,  15 
Soret's  band,  191 
Soup,  60 

Souring  of  milk,  56,  63 
Specific  gravity  of  blood,  222  ;  of  milk, 

49,  53  ;  of  urine,  141,  144 
Specific  rotatory  power,  229 
Spectra,  189 ;    of   haemoglobin  and   its 

derivatives,    189;     photographic,    of 

metheemoglobin,  190, 191  ;  oxyheemo- 

globin,  and  haemoglobin,  190,  191 ;  of 

urinary  pigments,  215 
Spectro-polarimeter,  229 
Spectroscope,  115,  116,  117 
Spectrum,  119,  189,  215 
Spermatozoa,  41 
Starch,   4,   13,   20,   65,    93,    95,    238; 

soluble,  21 ;  action  of  malt  on,  179  ; 

digestion    of,   68,   70;    test  for,   20, 

171 
Starling    and    Bayliss    on    pancreatic 

secretion,  83 
Starling  on  osmotic  pressure  of  proteins, 

243 
Steapsin,  25,  65,  78,  80,  82,  85  ;    action 

of,  78,  82 
Stearic  acid,  24 
Stearin,  23,  55 
Steatolytic  ferments,  65 


Stein's  method  of    making  oxyheemo- 

globin  crystals,  112 
Stellar  phosphates,  156 
Stercobihn,  92,  94,  95,  143 
Stewart,  G.  N.,  dietary,  51,  52 
Stirling  on  preparation  of  pure  oxyhsemo- 

globin,  191 
Stokes's  fluid,  115,  188 
Stomach,  glands  of,  71,  72 
Strontium,  8 
Sturine,  42 
Sublingual  gland,  67 
Submaxillary  gland,  67,  68,  69 
Succus  entericus,  84 
Sucroses,  16 
Sudan  III.  stain,  24 
Sugar,  4,  9,  13, 17-20,  87,  237;  cane,  13, 

18,  79 ;  in  blood,  110  ;  in  urine,  167, 

168  ;  in  muscle,  18  ;    tests  for,  166, 

174 
Sugar-maple,  18 
Sulphates  of  urine,  141, 153 ;  estimation 

of,  208  ;  tests  for,  141 
Sulphonal  poisoning,  214 
Sulphur,  3,  8  ;  tests  for,  10 
Suprarenal  gland,  removal  of,  87 
Suspending  medium,  23 
Sweetbreads,  160 
Swim-bladder  of  fishes,  131 
Symbols  and  atomic  weights,  8 
Syntonin,  30,  48,  62,  75 


Tannin,  61,  182 

Tapetum,  160 

Tartaric  acid,  144 

Taurine,  91,  94 

Tauro-carbamic  acid,  94 

Taurocholate  of  soda,  90,  91 

Taurocholic  acid,  91 

Tchistovitch's  experiment,  125 

Tea,  60,  61,  159 

Teichmann's  crystals,  113 

Tendo-mucoid,  45 

Tendon,  30 

Tension  in  fluids,  measurement  of,  132 

Tension  of  aqueous  vapour,  8  ;  of  gases, 

128,  130,  131 
Tension,   relation    of,   to    composition, 

132 
Testis,  196  ;  removal  of,  87 
Tetrapeptides,  36 
Tetroses,  16 
Theine,  60 

Theobromine,  60,  159  ' 
TheophyUine,  159 
Thermometric  scales,  7 
Thrombin,  104,  105,  106,  108 
Thrombogen,  105,  1^,  107,  108 


INDEX 


257 


Thrombo-kinase,    105,    106,    107,    108, 

196 
Thrombosis,  105 
Thudichum  on  urochrome,  213 
Thymine,  34 
Thymus,  159,  196 
Thyroid  gland,  87  ;  removal  of,  87 
Tin,  8 

Tissue-fibrinogens,  196 
Tissue  metabolism,  149 
Tissue  respiration,  126,  134,  136 
Tomes  on  enamel,  43 
Tooth,  43 
Topfer's  test,  185 
Torricellian  vacuum,  114 
Torula  ureae,  145 
Torulffi,  63,  145 
Toxins,  122,  123 
Transitory  giucosuria,  167 
Triacetin,  25 
Triatomic  alcohol,  25 
Trichloracetic  acid  as  a  precipitant  of 

proteins,  183 
Trimethyl-xanthine,  159 
Triolein,  25 
Trioses,  16 
Trioxy-purine,  159 
Tripalmitin,  25 
Tripeptides,  36 
Triple  phosphate,  156,  165 
Tristearin,  25 
Trivalent  elements,  236 
Trommer's  test,  13,  17,  18,  19,  20 
Tropffiolin  test,  185 
Trypsin,  65,  80,  81,  82,  84,  97  ;  action 

of,  81 
Trypsinogen,  80,  84,  186 
Tryptophan,  33,  34,  35,  81,   187;  test 

for,  187 
Tungsten,  8 
Tunicates,  21 
Tyrosine,  33,  34,  35,  42,  80,  81,  84,  86, 

165,  187  ;  crystals,-86  ;  in  urine,  165  : 

tests  for,  187  D"»f, 


tu 


XJffelmann's  reaction,  185 

Umbilical  cord,  30 

Uncrystallisable  sugar,  17 

Unorganised  ferments,  64,  66,  68 

Uracil,  46 

Uraemia,  147 

Uranium  acetate  in  estimation  of  phos- 
phates, 207 

Uranium  nitrate  in  estimation  of  phos- 
phates, 207 

Urate,  acid  ammonium,  deposit  of,  163 ; 
acid  sodium,  deposit  of,  163 

Urates,  156,  163,  165,  168,  214 


Urea,  4,  12,  30,  32,  87,  94,  97,  98,  110, 
141,  145-151,  161,  173,  237;  com- 
position and  compounds,  146 ; 
crystals  of,  145 :  decomposition  of, 
66 ;  estimation  of,  204 ;  mode  and 
site  of  formation,  147  ;  preparation 
of,  205  ;  quantity  excreted,  143  ;  tests 
for,  141,  145 ;  where  formed,  147 

Urea  nitrate,  146,  156 ;  preparation  of, 
156 

Urea  oxalate,  146,  156 ;  preparation  of, 
156 

Uric  acid,  110,  145,  156,  157-161,  159, 
160,  165,  173  ;  crystals  of,  157 ;  esti- 
mation of,  158,  210 ;  origin  of,  158 ; 
preparation  of,  157,  210 ;  tests  for, 
156,  157 

Uricolytic  ferment,  161 

Urina  potus,  144 

Urinary  deposits,  162 

Urine,  141 ;  composition  of,  144,  145, 
173  ;  inorganic  constituents  of,  153, 
173  ;  pigments  of,  143,  213 ;  tests  for 
abnormal  constituents  of,  174  ;  tests 
for  constituents,  142,  156 

Urinometer,  141,  144 

Urobilin,  92,  93, 143, 169,  213,  214, 216 ; 
absorption  bands  of,  215 

Urobilinogen,  143,  213 

Urochrome,     143,    213  ;     uro-erythrin, 
156,  214  ;  absorption  bands  of,  215 
j    Urorosein,   216 ;  absorption   bands   of, 
i        215 


Valeric  acid,  24,  32,  85 

Van 't  Hoff  on  polarisation  of  light,  231 ; 
hypothesis,  240 

Vegetable  acids,  4,  144  ;  food,  composi- 
tion of,  58  ;  parchment,  37 

Vegetables,  composition  of,  58 ;  green, 
61 

Velocity  of  chemical  reactions,  67,  82 

Venous  blood,  gases  of,  127 

Villus,  section  of,  98,  99 

Vinegar,  63 

Vital  action,  242 

Vitellin,  38,  44,  56,  58,  75 

Vitellose,  75 

Vitreous  humour,  30 

Voit's  diet,  51,  146 

Voluntary  muscle,  199 


W 


Waller's  modification  of  Zuntz's  gas 

apparatus,  234 
Warm  bath,  29 
Water  in  protoplasm,  3,  4  ;  density  of,  8 

S 


258 


ESSENTIALS   OF  CHEMICAL  PHYSIOLOGY 


Wave  theory  of  light,  222 

Weights  and  measures,  6,  7 

Whartonian  jelly,  30 

Whetstones,  156 

Whey,  49,  54  ;  salted,  49 

Whey  protein,  54 

White  blood  corpuscles,  110,  196 

White  of  egg,  3,  27,  29,  37,  38,  43,  45, 

62,  78,  180 
Whole  flour,  57,  58 
Widal's  reaction,  124 
Witte's  peptone,  182 
Wohler,  preparation  of  urea,  145 
Wooldridge  on  tissue-fibrinogen,  46, 196 
'  Worth'  of  corpuscles,  222 


Xanthine,  47,  80,  110,  169-161 
Xanthine  group,  46,  159 


Xanthoproteic  test,  27,  39 
Xylose,  178 


Yeast,  action  of,  17,  19,  66  ;    cells,  63  ; 

in  bread-making,  58 ;  in  testing  for 

sugar,  169 
Yellow  lipochrome,  200 
Yolk  of  eggs,  56 


Zein,  35,  48 
Zeiss's  polarimeter,  228 
Zinc,  8 

Zuntz's  gas  apparatus,  234 
Zymogen,  70,  72,  80, 105,  187  ;  granules, 
187 


PIIINTKD  BY 

SPOrnSWOODK   and   CO,    ltd.,    .\I:\V   STIlliKT   SQUAllK 

LUXDON 


A  LIST  OF  WORKS  ON 

MEDICINE,   SURGERY  AND 

GENERAL  SCIENCE 


CONTENTS 


PAGE 

BACTERIOLOGY 12 

BIOLOGY       8 

CHEMISTRY             14 

HEALTH  AND  HYGIENE          11 

MEDICINE 2 

MISCELLANEOUS            13 

OPTICS           13 

PHOTOGRAPHY 13 

PHYSIOLOGY          8 

SURGERY     2 

TEXT-BOOKS  OF  PHYSICAL  CHEMISTRY         16 

VETERINARY  MEDICINE         ...         ...        ' 8 

ZOOLOGY      8 


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ISSS.     Svo,  7s.  U. 
OCCASIONAL  PAPEES  ON  MEDICAL  SUBJECTS,  1855-1896. 

Svo,  12s. 

DOCKRELL.      AN   ATLAS   OF  DEEMATOLOGY :   showing  the 

Appearances,  Clinical  and  Microscopical,  Normal  and  Abnormal,  of  Condi- 
tions of  the  Skin.  60  Coloured  Plates  and  Descriptive  Letterpress.  By 
MORGAN  DOCKRELL,  M.A.,  M.D.  (Dub.  Univ.),  Senior  Physician  and 
Chesterfield  Lecturer  on  Dermatology  to  St.  John's  Hospital  for  Diseases- 
of  the  Skin.     Fcp.  folio,  50s.  net. 

* ^*  The  plate  showing  the  clinical  appearance  of  each  disease  appears 
on  the  same  page  with  that  displaying  the  microscopical.  The  descriptive 
letterpress  in  each  case  is  as  brief  as  possible. 

FOWLER  AND  GODLEE.    THE  DISEASES  OF  THE  LUNGS. 

By  JAMES  KINGSTON  FOWLER,  M.A.,  M.D.,  F.R.C.P.,  Physician  to 
the  Middlesex  Hospital  and  to  the  Hospital  for  Consumption  and  Diseases 
of  the  Chest,  Brompton,  etc.  ;  and  RICKMAN  JOHN  GODLEE,  M.S., 
F.R.C.S.,  Honorary  Surgeon-in-Ordinary  to  His  Majesty,  Fellow  and 
Professor  of  Clinical  Surgery,  University  College,  London,  etc.  With. 
160  Illustrations.     Svo,  25s. 


MESSRS.  LONGMANS'  WORKS  ON  MEDICINE,  SURGERY,  ETC.     5 

MEDICINE,  SURGERY,  RTC— continued. 
GARROD.    THE  ESSENTIALS  OF  MATEEIA  MEDICA  AND 

THERAPEUTICS.        By  Sir  ALFRED  BARING  GARROD,  M.D., 

F.R.S.,  etc.  ;  Consulting  Physician  to  King's  College  Hospital;  late  Vice- 
President  of  the  Royal  College  of  Physicians.  Fourteenth  Edition,  Re- 
vised and  Edited,  under  the  Supervision  of  the  Author,  by  NESTOR 
TIRARD,  M.D.  Lond.,  F.R.C.P.,  Professor  of  Materia  Medica  and  Thera- 
peutics in  King's  College,  London,  etc.     Crown  8vo,   12s.  6^. 

GOODSALL  AND  MILES.    DISEASES  OF  THE  ANUS  AND 

EECTUM.  By  D.  H.  GOODSALL,  F.R.C.S.,  late  Senior  Surgeon 
Metropolitan  Hospital,  Senior  Surgeon  St.  Mark's  Hospital ;  and  W. 
ERNEST  MILES,  P.R.C.S.,  Assistant  Surgeon  to  the  Cancer  Hospital, 
Surgeon  (out-patients)  to  the  Gordon  Hospital,  etc.      (In  Two  Parts). 

Part  I. — Anatomy  of  the  Ano-rectal  Region — General  Diagnosis — Abscess — 
Ano-rectal  Fistula  —  Recto-urethral,  Recto-vesical  and  Recto-vaginal 
Fistulas — Sinus  over  the  Sacro-coccygeal  Articulation — Fissure — Haemorr- 
hoids (External  and  Internal).     With  91  Illustrations.     8vo,  7s.  6d.  net. 

Part  II. — Prolapse  of  the  Rectum — Invagination  of  the  Rectum — Ulceration 
— Stricture  of  the  Anus  and  of  the  Rectum — Malignant  Growths  of  the 
Anus  and  Rectum — Benign  Tumours  of  the  Anus  and  Rectum — Foreign 
Bodies  in  the  Rectum — Pruritus  Ani — Syphilis  of  the  Anus  and  Rectum. 
With  44  Illustrations.     8vo,  6s.  net. 

GRAY.     ANATOMY,    DESCRIPTIVE     AND     SURGICAL.      By 

HENRY  GRAY,  F.R.S.,  late  Lecturer  on  Anatomy  at  St.  George's 
Hospital  Medical  School.  The  Sixteenth  Edition  Enlarged,  edited  by  T. 
PICKERING  PICK,  F.R.C.S.,  Consulting  Surgeon  to  St.  George's  Hospital, 
etc.,  and  by  ROBERT  HOWDEN,  M.A.,  M.B.,  CM.,  Professor  of  Anatoiny 
in  the  University  of  Durham,  etc.  With  811  Illustrations,  a  large  proportion 
of  which  are  coloured,  the  Arteries  being  coloured  red,  the  Veins  blue,  and  the 
Nerves  yellow.  The  attachments  of  the  muscles  to  the  bones,  in  the  section 
on  Osteology,  are  also  shown  in  coloured  outline.     Royal  Bvo,  32s.  net. 

HARE.    THE  FOOD  FACTOR  IN  DISEASE  :   Being  an  investiga- 

tion  into  the  humoral  causation,  meaning,  mechanism  and  rational  treat- 
ment, preventive  and  curative,  of  the  Paroxysmal  Neuroses  (migraine, 
asthma,  angina  pectoris,  epilepsy,  etc.),  bilious  attacks,  gout,  catarrhal 
and  other  affections,  high  blood-pressure,  circulatory,  renal  and  other 
degenerations.  By  FRANCIS  HARE,  M.D.,  late  Consulting  Physician  to 
the  Brisbane  General  Hospital ;  Visiting  Physician  at  the  Diamantina 
Hospital  for  Chronic  Diseases,  Brisbane  ;  Inspector-General  of  Hospitals 
for  Queensland.     2  vols.     Medium  Bvo,  30s.  net. 

LACK.      THE   DISEASES    OF   THE    NOSE    AND    ITS    AC- 
CESSORY   SINUSES.       By  H.   LAMBERT  LACK,   M.D.   Lond., 

F.R.C.S.  Eng.,  Surgeon  to  the  Throat  Department,  London  Hospital; 
Surgeon  to  the  Hospital  for  Diseases  of  the  Throat,  Golden  Square ; 
Lecturer  on  Diseases  of  the  Throat,  London  Hospital  Medical  College 
(University  of  London).     With  124  Illustrations.     Medium  8vo,  25s. 

LANG.     THE     METHODICAL     EXAMINATION      OF     THE 

EYE.  Being  Part  I.  of  a  Guide  to  the  Practice  of  Ophthalmology  for 
Students  and  Practitioners.  By  WILLIAM  LANG,  F.R.C.S.  Eng.,  Surgeon 
:.o  the  Royal  London  Ophthalmic  Hospital,  Moorfields,  etc.  With  15 
illustrations.     Crown  8vo,  3s.  6d. 


6    MESSRS.  LONGMANS'  WORKS  ON  MEDICINE,  SURGERY,  ETC. 


MEDICINE,  SURGERY,  RTC—cofitinued. 
LUFF.       TEXT -BOOK     OF    FOEENSIC     MEDICINE     AND 

TOXICOLOGY.  By  ARTHUR  P.  LUFF,  M.D.,  B.Sc.  Lond., 
Physician  in  Charge  of  Out-Patients  and  Lecturer  on  Medical  Jurisprudence 
and  Toxicology  in  St.  Mary's  Hospital ;  Examiner  in  Forensic  Medicine  in 
the  University  of  London  ;  External  Examiner  in  Forensic  Medicine  in  the 
Victoria  University ;  Official  Analyst  to  the  Home  Office.  With  13  full- 
page  Plates  (1  in  colours)  and  33  Illustrations  in  the  Text.  2  vols.,  Crown 
8vo,  24s. 

PROBYN-WILLIAMS.       A    PEACTICAL    GUIDE    TO    THE 
ADMIN ISTEATION     OF     ANESTHETICS.        By    R.    j. 

PROBYN-WILLIAMS,  M.D.,  Anaesthetist  and  Instructor  in  Ansesthetics 
at  the  London  Hospital ;  Lecturer  in  Ansesthetics  at  the  London  Hospital 
Medical  College,  etc.     With  34  Illustrations.     Crown  8vo,  4s.  6^.  net. 


QUAIN.     QUAIN'S     (JONES)     ELEMENTS    OF    ANATOMY. 

The  Tenth  Edition.  Edited  by  EDWARD  ALBERT  SCHAFER,  F.R.S., 
Professor  of  Physiology  in  the  University  of  Edinburgh  ;  and  GEORGE 
DANCER  THANE,  Professor  of  Anatomy  in  University  College,  London. 

*^*  The  several  parts  of  this  work  form  complete  Text-Books  of  their 
RESPECTIVE  subjects.     They  can  be  obtained  separately  as  follows  : — 

Vol.  I.,  Part  I.  EMBEYOLOGY.  By  E.  A.  SCHAFER,  F.R.S.  With 
200  Illustrations.     Royal  8vo,  9s. 

Vol.  I.,  Part  II.     GENEEAL    ANATOMY    OE    HISTOLOGY. 

By  E.  A.  SCHAFER,  F.R.S.     With  491  Illustrations.     Royal  8vo,  12s.  6d. 

Vol.  II.,  Part  I.  OSTEOLOGY  —  AETHEOLOGY.  By  G.  D. 
THANE.     With  224  Illustrations.     Royal  8vo,  lis. 

Vol.  II.,  Part  II.  MYOLOGY  —  ANGEIOLOGY.  By  G.  D. 
THANE.     With  199  Illustrations.     Royal  Bvo,  16s. 

Vol.  III.,  Part  I.  THE  SPINAL  COED  AND  BEAIN.  By  E.  A. 
SCHAFER,  F.R.S.     With  139  Illustrations.     Royal  Bvo,  12s.  6d. 

Vol.  IIL,  Part  II.  THE  NEEVES.  By  G.  D.  THANE.  With  102 
Illustrations.     Royal  8vo,  9s. 

Vol.  III.,  Part  III.  THE  OEGANS  OF  THE  SENSES.  By  E.  A. 
SCHAFER,  F.R.S.     With  178  Illustrations.     Royal  Bvo,  9s. 

Vol.  III.,  Part  IV.  SPLANCHNOLOGY.  By  E.  A.  SCHAFER, 
F.R.S.,  and  JOHNSON  SYMINGTON,  M.D.  With  337  Illustrations. 
Royal,  8vo,  16s. 

Appendix.  SUPEEFICIAL  AND  SUEGICAL  ANATOMY.  By 
Professor  G.  D.  THANE  and  Professor  R.  J.  GODLEE,  M.S.  With  29 
Illustrations.     Royal  Bvo,  6s.  6d. 


MESSRS.  LONGMANS'  WORKS  ON  MEDICINE,  SURGERY,  ETC.    7 

MEDICINE,  SURGERY,  ETC.— continued. 

QUAIN.  QUAIN'S  (Sie  EICHARD)  DICTIONARY  OF  MEDI- 
CINE. By  Various  Writers.  Edited  by  H.  MONTAGUE  MURRAY, 
M.D.,  F.R.C.P.,  Joint  Lecturer  on  Medicine,  Charing  Cross  Medical  School, 
and  Physician  to  Charing  Cross  Hospital,  and  to  the  Victoria  Hospital  for 
Children,  Chelsea  ;  Examiner  in  Medicine  to  the  University  of  London. 
Assisted  by  JOHN  HAROLD,  M.B.,  B.Ch.,  B.A.O.,  Physician  to  St. 
John's  and  St.  Elizabeth's  Hospital,  and  Demonstrator  of  Medicine  at 
Charing  Cross  Medical  School,  and  W.  CECIL  BOSANQUET,  M.A., 
M.D.,  F.R.C.P.,  Assistant  Physician,  Charing  Cross  Hospital,  etc.  Third 
and  Cheaper  Edition,  largely  Rewritten,  and  Revised  throughout.  With 
21  Plates  (14  in  Colour)  and  numerous  Illustrations  in  the  Text.  8vo, 
21s.  net.,   buckram  ;    30s.  net.,  half-morocco. 


SCHAFER.      THE  ESSENTIALS  OF  HISTOLOGY:  Descriptive 

and  Practical.  For  the  Use  of  Students.  By  E.  A.  SCHAFER,  F.R.S., 
Professor  of  Physiology  in  the  University  of  Edinburgh.  With  553  Illus- 
trations some  of  which  are  Coloured.     8vo,  10s.  6^.  net. 


SMALE   AND   COLYER.       DISEASES  AND   INJURIES  OF 

THE  TEETH,  including  Pathology  and  Treatment.  By  MORTON 
SMALE,  M.R.C.S.,  L.S.A.,  L.D.S.,  Dental  Surgeon  to  St.  Mary's  Hospital, 
Consulting  Dental  Surgeon,  Dental  Hospital  of  London,  etc. ;  and  J.  F. 
COLYER,  L.R.C.P.,  M.R.C.S.,  L.D.S.,  Dental  Surgeon  to  Charing  Cross 
Hospital  and  to  the  Dental  Hospital  of  London,  Dean  of  the  School,  Dental 
Hospital  of  London.  Second  Edition  Revised  and  Enlarged  by  J.  F. 
COLYER.     With  640  Illustrations.     Large  Crown  8vo,  21s.  net. 


SMITH  (H.   F.).      THE    HANDBOOK   FOR    MID  WIVES.     By 

HENRY  FLY   SMITH,  B.A.,   M.B.   Oxon.,  M.R.C.S.     Second  Edition. 
With  41  Woodcuts.     Crown  Bvo,  5s. 


STEVENSON.  WOUNDS  IN  WAR  :  the  Mechanism  of  their 
Production  and  their  Treatment.  By  Surgeon-General  W.  F.  STEVENSON, 
C.B.  (Army  Medical  Staff),  B.A.,  M.B.,  M.Ch.  Dublin  University ;  Professor 
of  Military  Surgery,  Royal  Army  Medical  College,  London.  With  127 
Illustrations.     Bvo,  15s.  net. 


TAPPEINER.    INTRODUCTION   TO  CHEMICAL   METHODS 

OF  CLINICAL  DIAGNOSIS.  By  Dr.  H.  TAPPEINER,  Professor 
of  Pharmacology  and  Principal  of  the  Pharmacological  Institute  of  the 
University  of  Munich.  Translated  from  the  Sixth  German  Edition,  with 
an  Appendix  on  Micro-Biological  Methods  of  Diagnosis,  by  EDMOND  J. 
McWEENEY,  M.A.,  M.D.  Royal  Univ.  of  Ireland,  L.R.C.P.I.,  etc. 
With  22  Illustrations.     Crown  8vo,  3s.  6d. 


8    MESSRS.  LONGMANS'  WORKS  ON  MEDICINE,  SURGERY,  ETC. 


VETERINARY  MEDICINE,  ETC. 

FITZWYGRAM.     HOESES    AND    STABLES.      By  Lieutenant- 

General  Sir  F.  FITZWYGRAM,  Bart.     With  56  pages  of  Illustrations. 
8vo,  3s.  net. 

HAYES.   TEAINING  AND  HORSE  MANAGEMENT  IN  INDIA. 

With  Hindustanee  Vocabulary.     By  M.  HORACE   HAYES,  F.R.C.V.S. 
(late  Captain,  "  The  Buffs  ").     With  Portrait.     Crown  8vo,  8s.  net. 

STEEL— WORKS  by  JOHN  HENRY  STEEL,  F.R.C.V.S.,  F.Z.S., 
A.  V.D.,  late  Professor  of  Veterinary  Science  and  Principal  of  Bombay  Veterinary 
College. 

A  TREATISE  ON  THE  DISEASES  OF  THE  DOG ;  being 
a  Manual  of  Canine  Pathology.  Especially  adapted  for  the  use  of 
Veterinary  Practitioners  and  Students.  With  88  Illustrations.  8vo. 
10s.  bd. 

A  TREATISE  ON  THE  DISEASES  OF  THE  OX ;  being  a 
Manual  of  Bovine  Pathology.  Especially  adapted  for  the  use  of  Veterinary 
Practitioners  and  Students.     With  2  Plates  and  117  Woodcuts.     8vo,  15s. 

A  TREATISE  ON  THE  DISEASES  OF  THE  SHEEP ;  being 
a  Manual  of  Ovine  Pathology  for  the  use  of  Veterinary  Practitioners  and 
Students.     With  Coloured  Plate  and  99  Woodcuts.     8vo,  12s. 

YOUATT— WORKS  by  WILLIAM  YOUATT. 

THE  HORSE.  Revised  and  Enlarged  by  W.  WATSON,  M.R.C.V.S. 
With  52  Wood  Engravings.     8vo,  7s.  6^. 

THE   DOG.     Revised  and  Enlarged.    With  33  Wood  Engravings.    8vo,  6s. 


PHYSIOLOGY,  BIOLOGY,  ZOOLOGY,  ETC. 

ASH  BY.  NOTES  ON  PHYSIOLOGY  FOR  THE  USE  OF 
STUDENTS  PREPARING  FOR  EXAMINATION.  By 
HENRY  ASHBY,  M.D.  (Lond ),  F.R.C.P.,  Physician  to  the  General 
Hospital  for  Sick  Children,  Manchester  ;  Lecturer  and  Examiner  in 
Diseases  of  Children  in  the  Victoria  University.  With  148  Illustrations, 
18mo,  5s. 

BARNETT.     THE  MAKING  OF  THE  BODY:  a  Children's  Book 

on   Anatomy   and   Physiology.      By   Mrs.    S.    A.    BARNETT.     With  113 
Illustrations.     Crov^rn  8vo,  Is.  9d. 

BEDDARD.      ELEMENTARY     PRACTICAL    ZOOLOGY.      By 

FRANK  E.  BEDDARD,  M.A.  (Oxon.).      With  93  Illustrations.      Crown 
Svo,  2s.  6d. 

BIDGOOD.      A    COURSE    OF    PRACTICAL    ELEMENTARY 

BIOLOGY.      By  JOHN  BIDGOOD,  B.Sc,  F.L.S.     With  226  Illustra- 
tions.    Crown  Svo,  4s.  6d. 


MESSRS.  LONGMANS'  WORKS  ON  MEDICINE,  SURGERY,  ETC.     9 

PHYSIOLOGY,  BIOLOGY,  ZOOLOGY,  RTC— continued. 

BOSE.—^^ORKS  by  JAGADIS  GHUNDER  ROSE,  M.A.  {Gantah.),  D.Sc. 
(Land.),  Professor-,  Preside7icy  College,  Calcutta. 

RESPONSE  IN  THE  LIVING  AND   NON-LIVING.      With 
117  Illustrations.     8vo,  10s.  &d. 

PLANT  RESPONSE  AS  A  MEANS  OF  PHYSIOLOGICAL 

INVESTIGATION.       with  278  Illustrations.      Svo,  21s. 
ELECTRO-PHYSIOLOGY  OF  PLANTS.  [In  the  press. 

BRODIE.  THE  ESSENTIALS  OF  EXPERIMENTAL  PHY- 
SIOLOGY. For  the  use  of  Students.  By  T.  G.  BRODIE,  M.D., 
Lecturer  on  Physiology,  St.  Thomas's  Hospital  Medical  School.  With 
2  Plates  and  177  Illustrations  in  the  Text.     Crown  Svo,  6s.  Qd. 

CHAPMAN.     THE   FORAMINIFERA :    AN  INTRODUCTION 

TO  THE  STUDY  OF  PROTOZOA.  By  FREDERICK  CHAP- 
MAN, A.L.S.,  F.R.M.S.,  formerly  Assistant  in  the  Geological  Laboratory 
of  the  Royal  College  of  Science  ;  Palaeontologist  to  the  National  Museum, 
Melbourne.     With  14  Plates  and  42  Illustrations  in  the  Text.     Svo,  9s.  net. 

FURNEAUX.     HUMAN    PHYSIOLOGY.      By  w.  FURNEAUX, 

F.R.G.S.     With  223  Illustrations.     Crown  Svo,  2s.  6d. 

HALLIBURTON.-^rOjR^.S     hy     W.    D.     HALLIBURTON,    M.D., 

F.R.S.,  F.R.C.P..  Professor  of  Physiology  in.  King's  College,  London. 

A     TEXT-BOOK     OF     CHEMICAL     PHYSIOLOGY     AND 

PATHOLOGY.      With  104  Illustrations.     Svo,  28s. 
THE  ESSENTIALS  OF  CHEMICAL  PHYSIOLOGY.    For  the 

Use  of  Students.     With  S3  Illustrations.     Svo,  4s.  6d.  net. 

HUDSON  AND  GOSSE.      THE   ROTIFERA   OR    "WHEEL 

ANIMALCULES".  By  C.  T.  HUDSON,  LL.D.,  and  P.  H.  GOSSE, 
F.R.S.  With  30  Coloured  and  4  Uncoloured  Plates.  In  6  Parts.  4to,  price 
10s.  6d.  each ;  Supplement,  12s.  6d.  Complete  in  Two  Volumes,  with 
Supplement,  4to,  £4  4s. 

*^*  The  Plates  in  the  Supplement  contain  figures  of  almost  all  the  Foreign 
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original  publication  of  Vols.  I.  and  II. 

LLOYD.  THE  TEACHING  OF  BIOLOGY  IN  THE  SECON- 
DARY SCHOOL.  By  FRANCIS  E.  LLOYD,  A.M.,  and  MAURICE 
A.  BIGELOW,  Ph.D.,  Professors  in  Teachers'  College,  Columbia  Univer- 
sity.    Crown  Svo,  6s.  net. 

JVIACALISTER.— W^O/^j^S'  by  ALEXANDER  MAGALISTER,  M.D. 

AN    INTRODUCTION    TO    THE    SYSTEMATIC    ZOOLOGY 
AND     MORPHOLOGY     OF     VERTEBRATE     ANIMALS. 

With  41  Diagrams.     Svo,  10s.  e,d. 

ZOOLOGY  OF  THE  INVERTEBRATE  ANIMALS,     with  77 

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ZOOLOGY    OF    THE    VERTEBRATE    ANIMALS,      with  59 
Diagrams.     Fcp.  Svo,  Is.  6d. 


10  MESSRS.  LONGMANS'  WORKS  ON  MEDICINE,  SURGERY,  ETC. 


PHYSIOLOGY,  BIOLOGY,  ZOOLOGY,  RTC— continued, 

MACDOUGALL.-WORKS  by  DANIEL  TREMBLY MAGDOUGALL, 

Ph.  J). 

TEXT-BOOK  OF  PLANT  PHYSIOLOGY,      with  159  lllustia- 

tions.      8vo,  7s.  6d.   net, 

ELEMENTAEY  PLANT  PHYSIOLOGY,      with  108  Illustrations. 
Crown  8vo,  3s. 

MOORE.       ELEMENTAEY     PHYSIOLOGY.       By    BENJAMIN 

MOORE,  D.Sc,  Professor  of  Biological  Chemi'^try  in  the  University  of 
Liverpool.      Crown  8vo,  3s.  M. 

MORGAN.  ANIMAL  BIOLOGY.  An  Elementary  Text-Book.  By 
C.  LLOYD  MORGAN,  F.R.S.,  Principal  of  University  College,  Bristol. 
With  numerous  Illustrations.     Crown  8vo,  8s.  6d. 

SCHA'='ER.     DIEECTIONS    FOE    CLASS    WOEK    IN    PEAC- 

TICAL  PHYSIOLOGY  :  Elementary  Physiology  of  Muscle  and  Nerve 
and  of  the  Vascular  and  Nervous  Systems.  By  E.  A.  SCHAFER,  LL.D., 
F.R.S.,  Professor  of  Physiology  in  the  University  of  Edinburgh.  With  48 
Diagrams.     8vo,  3s.  net. 

THORHJOU— WORKS  by  JOHN  THORNTON,  M.A. 

HUMAN     PHYSIOLOGY.       with  284  Illustrations,  some   of  which 
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\^MA.ER.-WORKS  by  AUGUSTUS  D.  WALLER,  M.D.,  F.R.S., 
Hon.  LL.D.  JMin.,  Lecturer  on  Physiology  at  St.  Mary's  Hospital  Medical 
School,  London  ;  late  External  Examiner  at  the  Victorian  University. 

AN   INTEODUCTION   TO    HUMAN    PHYSIOLOGY.     With 

314  Illustrations.     8vo,  18s. 
LECTUEES  ON  PHYSIOLOGY. 

First  Series. — On  Animal  Electricity.     8vo,  5s.  net. 

WALLER  AND  SYMES.  EXEECISES  IN  PEACTICAL  PHY- 
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Part  II. — Exercises  and  Demonstrations  in  Chemical  and  Physical  Physiology. 
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Part   III.— Physiology   of    the   Nervous    System,   Electro-Physiology.      By 
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MESSBS.  LONGMANS'  WOBKS  ON  MEDICINE,  SURGERY,  ETC.  11 


HEALTH  AND  HYGIENE,  ETC. 

ASH  BY.  HEALTH  IN  THE  NUESEEY.  By  HENRY  ASHBY, 
M.D.,  F.R.C.P.,  Physician  to  the  General  Hospital  for  Sick  Children, 
Manchester ;  Lecturer  and  Examiner  in  Diseases  of  Children  in  the- 
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BRODRIBB.     MANUAL  OF   HEALTH  AND  TEMPEEANCE. 

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W.  RUTHVEN  PYM,  M.A.     Crown  8vo,  Is.  6d. 

BUCKTON.       HEALTH    IN    THE    HOUSE.       By  Mrs.   c.   M. 

BUCKTON.     With  41  Woodcuts  and  Diagrams.     Crown  8vo,  2s. 

CORFIELD.     THE  LAWS  OF  HEALTH.     By  w.  H.  CORFIELD,. 

M.A.,  M.D.     Fcp.  8vo,  Is.  6d. 

FURNEAUX.  ELEMENTAEY  PEAOTICAL  HYGIENE.  Sec- 
tion I.  By  WILLIAM  S.  FURNEAUX.  With  146  Illustrations.  Crown 
8vo  2s.  6d. 

NOTTER  AND  FIRTH.— WORKS  by  J.  L.  NOTTER,  M.A.,  M.D.,  and 

R.  H.  FIRTH,  F.R.C.S. 

HYGIENE.     With  99  Illustrations.     Crown  8vo,  4s.  Qd. 

PEACTICAL    DOMESTIC    HYGIENE.        with    84    Illustrations. 
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POORE— WORKS  by  GEORGE    VIVIAN  POORE,  M.D.,  F.R.C.P. 

THE   EAETH   IN   EELATION   TO   THE   PEESEEVATION 
AND    DESTEUCTION    OF    CONTAGIA :    being   the   Milroy 

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HEALTH  :  a  Text-Book  for  Teachers  and  Students  in  Training.  By 
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ROBINSON.    THE  HEALTH  OF  OUE  CHILDEEN  IN  THE 

COLONIES  :  a  Book  for  Mothers.  By  LILIAN  AUSTEN  ROBIN- 
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12  MESSRS.  LONGMANS'  WORKS  ON  MEDICINE,  SURGERY,  ETC. 


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CURTIS.      THE    ESSENTIALS    OF   PEACTICAL    BACTEEI- 

OLOGY  :  an  Elementary  Laboratory  Work  for  Students  and  Practitioners. 
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FRANKLAND.       MICEO-OEGANISMS   IN   WATEE,    THEIE 
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PERCY  FRANKLAND,  Joint  Author  of  "  Studies  on  Some  New  Micro- 
organisms Obtained  from  Air".  With  2  Plates  and  numerous  Diagrams. 
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FRANKLAND.     BACTEEIA  IN  DAILY  LIFE.      By  Mrs.  PERCY 

FRANKLAND,  P.R.M.S.      Crown  8vo,  5s.   net. 


GOADBY.  THE  MYCOLOGY  OF  THE  MOUTH:  A  TEXT- 
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KLOCKER.      FEEMENTATION     OEGANISMS.      a  Laboratory 

Handbook.  By  ALB.  KLOCKER,  Assistant  in  the  Carlsberg  Laboratory, 
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Lecturer  in  the  University  of  Birmingham,  and  J.  H.  MILLAR,  F.I.C., 
formerly  Lecturer  in  the  British  School  of  Malting  and  Brewing,  and 
revised  by  the  Author.      With  146  Illustrations.     8vo,  12s.  net. 


PLIMMER.  THE  CHEMICAL  CHANGES  AND  PEODUCTS 
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OPTICS,  PHOTOGRAPHY,  ETC. 

ABNEY.     A  TEEATISE  ON  PHOTOGRAPHY.     By  Sir  WILLIAM 

DE  WIVELESLIE  ABNEY,  K.C.B.,  P.R.S.,  Principal  Assistant  Sec- 
retary of  the  Secondary  Department  of  the  Board  of  Education.  With 
134  Illustrations.     Crown  8vo,  5s. 

DRUDE.      THE  THEOEY   OF  OPTICS.      By  PAUL  DRUDE,  Pro- 

fessor  of  Physics  at  the  University  of  Giessen.  Translated  from  the- 
German  by  C.  RIBORG  MANN  and  ROBERT  A.  MILLIKAN,  Assistant 
Professors  of  Physics  at  the  University  of  Chicago.  With  110  Diagrams.. 
8vo,  15s.  net. 

GLAZEBROOK.     PHYSICAL  OPTICS.     By  R.  T.  glazebrook, 

M.A.,  F.R.S.,  Principal  of  University  College,  Liverpool.  With  183  Wood- 
cuts of  Apparatus,  etc.     Crown  8vo,  6s. 

VANDERPOEL.  COLOUR  PROBLEMS:  a  Practical  Manual  for 
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117  Plates  in  Colour.     Square  8vo,  21s.  net. 

WRIGHT.  OPTICAL  PROJECTION:  A  Treatise  on  the  Use  of  the- 
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WRIGHT,  Author  of  "  Light :  a  Course  of  Experimental  Optics  ".  Withi 
232  Illustrations.     Crown  8vo,  6s. 


MISCELLANEOUS. 

INQUIRY  (AN)  INTO  THE  PHENOMENA  ATTENDING^ 
DEATH  BY  DROWNING  AND  THE  MEANS  OF  PRO- 
MOTING RESUSCITATION  IN  THE  APPARENTLY 
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and  Chirurgical  Society.  With  2  Diagrams  and  26  Folding-out  Plates. 
8vo,  5s.  net, 

HOBART.     THE  MEDICAL  LANGUAGE  OF  ST.  LUKE.     By 

the  Rev.  WILLIAM  KIRK  HOBART,  LL.D.     8vo,  16s. 

PAGET.     MEMOIRS  AND  LETTERS  OF  SIR  JAMES  PAGET, 

Bart.,  F.R.S.,  D.C.L.,  late  Sergeant-Surgeon  to  Her  late  Majesty  Queen 
Victoria.  Edited  by  STEPHEN  PAGET,  F.R.C.S.  With  Portrait.  Svo^ 
6s.  net. 

POOLE.  COOKERY  FOR  THE  DIABETIt).  By  w.  H.  and 
Mrs.  POOLE.      With  Preface  by  Dr.  PAVY.      Fcap.  8vo,  2s.  6d. 


14  MESSRS.  LONGMANS'  WORKS  ON  MEDICINE,  SURGERY,  ETC. 


CHEMISTRY,  ETC. 

ARMITAGE.     A  HISTOEY  OF  CHEMISTEY.     By  F.  P.  armi- 

TAGE,  M.A.,  F.C.S.,  late  Exhibitioner  of  Magdalen  College,  Oxford, 
Assistant  Master  at  St.  Paul's  School.     Crown  8vo,  6.s. 

ARRHENIUS.       A  TEXT-BOOK  OF  ELEOTEO-CHEMISTRY. 

By  SVANTE  ARRHENIUS,  Professor  of  Physics  at  the  University  of 
Stockholm.  Translated  from  the  German  Edition  by  JOHN  McCRAE, 
Ph.D.      With  58  Illustrations.      Svo,  9s.  6d.  net. 

CROOKES.     SELECT  METHODS  IN  CHEMICAL  ANALYSIS 

(chiefly  inorganic).  By  Sir  W.  CROOKES,  F.R.S.  With  68  Illustrations. 
8vo,  21s,  net. 

FINDLAY.  PHYSICAL  CHEMISTEY  AND  ITS  APPLICA- 
TIONS IN  MEDICAL  AND  BIOLOGICAL  SCIENCE. 
Being  a  Course  of  Seven  Lectures  delivered  in  the  University  of  Bir- 
mingham. By  ALEX.  FINDLAY.  M.A.,  Ph.D.,  D.Sc,  Lecturer  on 
Physical  Chemistry,  University  of  Birmingham.     Royal  8vo,  2s.  net. 

GREGORY,     A  SHOET  INTEODUCTION  TO  THE  THEOEY 

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JVIENDELEEFF.     THE   PEINCIPLES   OF   CHEMISTEY.      By 

D.  MENDELEEFF.  Translated  from  the  Russian  (Seventh  Edition)  by 
GEORGE  KAMENSKY,  A.R.S.M.,  of  the  Imperial  Mint,  St.  Petersburg, 
and  Edited  by  THOMAS  H.  POPE,  B.Sc,  F.I.C.  With  110  Illustrations. 
2  vols.     8vo,  32s.  net 

MEYER.      OUTLINES     OF     THEOEETICAL     CHEMISTEY. 

By  LOTHAR  MEYER,  Professor  of  Chemistry  in  the  University  of 
Tubingen.  Translated  by  Professors  P.  PHILLIPS  BEDSON,  D.Sc, 
and  W.  CARLETON  WILLIAMS,  B.Sc     8vo,  9s. 

MUIR.     A  COUESE  OF  PEACTICAL  CHEMISTEY.     By  M.  M. 

P.  MUIR,  M.A.,  Fellow  and  Prselector  in  Chemistry  of  Gonville  and  Caius 

College,  Cambridge.     (3  Parts.) 
Part  I.     Elementary.     Crown  8vo,  4s,  6d. 
Part  11.     Intermediate.     Crown  8vo,  4s.  6d. 

NEV^TH.— WORKS  by  G.  S.  NEWTlf,  F.I.C,  F.C.S.,  Demomtiator  in  the 

Royal  (JoUejie  of  ,^cience,   London. 

CHEMICAL    LECTUEE     EXPEEIMENTS.       with  230  Illustra- 
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SMALLEE  CHEMICAL  ANALYSIS.     Crown  8vo,  2s. 
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ELEMENTAEY  PEACTICAL  CHEMISTEY.     with  108  lilustra- 
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PERKm.—  WORKii  by  F.  MOLLWO  PERKIN,  Ph.D.,  Head  of  the 
Chemistry  JJejMrtment,  Borough  Polytechnic  InstittUe,  London. 

QUALITATIVE    CHEMICAL    ANALYSIS    (OEGANIC   AND 
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PEACTICAL  METHODS  OF  ELECTEO-CHEMISTEY.     Svo, 
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RADCLIFFE  AND  SINNATT.  A  SYSTEMATIC  COUESE  OF 
PEACTICAL  OEGANIC  CHEMISTEY.   By  LIONEL  GUY  KAD- 

CLIFFE,  F.C.S.,  Lecturer  in  Organic  Chemistry,  Municipal  School  of 
Technology,  Manchester,  with  the  assistance  of  FRANK  STURDY  SIN- 
NATT, F.C.S.,  Lecturer  and  Demonstrator  in  Chemistry,  Municipal  School 
of  Technology,  Manchester.     Svo,  4s.  Qd. 

REYNOLDS.    EXPEEIMENTAL  CHEMISTEY  for  Junior  students. 
By  J.  EMERSON  REYNOLDS,  M.D.,  F.R.S.,  Professor  of   Chemistry, 
University  of  Dublin.     Fcap.  Svo,  with  numerous  Illustrations. 
Part  I. — Introductory,  Is.  Qd.     Part  III. — Metals  mid  Allied  Bodies,  3s.  Qd. 
Part  II. — Non-Metals,  2s.  6d.     Part  IV. — Chemistry  of  Carbon  Comjjounds,  4s. 

SMITH  AND  HALL.  THE  TEACHING  OF  CHEMISTEY 
AND  PHYSICS  IN  THE  SECONDAEY  SCHOOL.  By  ALEX- 
ANDER SMITH,  B.Sc,  Ph.D.,  Associate  Professor  of  Chemistry  in  the 
University  of  Chicago,  and  EDWIN  H.  HALL,  Ph.D.,  Professor  of  Physics 
in  Harvard  University.  With  21  Woodcuts,  Bibliographies,  and  Index. 
Ciown  Svo,  6s.  net. 

THORPE.       A    DICTIONAEY    OF    APPLIED    CHEMISTEY. 

By  T.  E.  THORPE,  C.B.,  D.Sc.  Vict.,  Ph.D.,  F.R.S.,  Principal  of 
Government  Laboratory,  London.  Assisted  by  Eminent  Contributors. 
3  vols.     Svo.      Vols.  I.  and  II.,  £2  2s.  each  ;  Vol.  III.,  £3  3s. 

TILDEN.  A  SHOET  HISTOEY  OF  THE  PEOGEESS  OF 
SCIENTIFIC  CHEMISTEY  IN  OUE  OWN  TIMES.  By 
WILLIAM  A.  TILDEN,  D.Sc.  Lond.,  D.Sc.  Dub.,  F.R.S.,  Fellow  of  the 
University  of  London,  Professor  of  Chemistry  in  the  Royal  College  of 
Science,  London.     Crown  Svo,  5s.  net. 

WATTS   (H.).      DICTIONAEY   OF   CHEMISTEY.     Revised   and 

entirely  Re-written  by  H.  FORSTER  MORLEY,  M.A.,  D.Sc,  Fellow 
of,  and  lately  Assistant-Professor  of  Chemistry  in.  University  College, 
London;  and  M.  M.  PATTISON  MUIR,  M.A.,  F.R.S.E.,  Fellow  and 
Prselector  in  Chemistry  of  Gonville  and  Caius  College,  Cambridge. 
Assisted  by  Eminent  Contributors.     4  vols.     Svo,  £5  net. 

WESTON.  A  SCHEME  FOE  THE  DETECTION  OF  THE 
MOEE  COMMON  CLASSES  OF  CAEBON  COMPOUNDS. 
By  FRANK  E.  WESTON,  B.Sc,  London  (Fiist  Class  Honours),  F.C.S., 
Lecturer  in  Chemistry  at  the  Polytechnic,  Regent  Street,  W.     Svo,  2s. 


16  MESSRS.  LONGMANS'  WORKS  ON  MEDICINE,  SURGERY,  ETC. 

CHEMISTRY,  RTC— continued. 

VJH\TELEY.— WORKS     hi  R.    L.    Whiteley,   F.I.G.,   Principal  of  the 

Municijjal  Science  School,  West  Broviwich. 
CHEMICAL  CALCULATIONS,     with  Explanatory  Notes,  Problems, 
and  Answers,  specially  adapted  for  use  in  Colleges  and  Science  Schools. 
With  a  Preface  by  Professor  F.  CLOWES,  D.Sc.  (Lond.),  F.I.C.     Crown 
8vo,  2s. 

OEGANIC   CHEMISTEY  :  the  Fatty  Compounds.     With  45  Illustra^ 
tions.     Crown  8vo,  3s.  &d. 


TEXT.BOOKS  OF  PHYSICAL  CHEMISTRY. 

Edited  by  Sir  WILLIAM  RAMSAY,  K.C.B.,  F.R.S. 

THE  PHASE  EULE  AND  ITS  APPLICATIONS.  By  ALEX. 
FINDLAY,  M.A.,  Ph.D.,  D.Sc,  Lecturer  and  Demonstrator  in  Chemistry, 
University  of  Birmingham.  With  134  Figures  in  the  Text,  together  with 
an  "  Introduction  to  the  Study  of  Physical  Chemistry"  by  Sir  WILLIAM 
RAMSAY,  K.C.B.,  F.R.S.,  Editor  of  the  Series.     Crown  8vo,  5s. 

A  PEACTICAL  INTEODUCTION  TO  CHEMISTEY.  intended 
to  give  a  Practical  acquaintance  with  the  Elementary  Facts  adn  Prin- 
ciples of  Chemistry.      With  25  Illustrations.      Crown  Svo,  2s. 

ELECTEO- CHEMISTEY.   PAET  L— GENEEAL  THEOEY. 

By  R.  A.  LEHFELDT,  D.Sc,  Professor  of  Physics  at  the  East  London 
Technical  College.  Including  a  Chapter  on  the  Relation  of  Chemical  Con- 
stitution to  Conductivity,  by  T.  S.  MOORE,  B.A.,  B.Sc,  Lecturer  in 
the  University  of  Birmingham.      Crown  Svo,  5s. 

ELECTEO  -  CHEMISTEY.  PAET  II.— APPLICATIONS  TO 
ELECTEOLYSIS,  PEIMAEY  AND  SECONDAEY  BAT- 
TEEIES,  ETC.  [In  preparation. 

CHEMICAL  STATICS  AND  DYNAMICS,  INCLUDING  THE 
THEOEIES  OF  CHEMICAL  CHANGE,  CATALYSIS,  AND 
EXPLOSIONS.  By  J.  W.  MELLOR,  D.Sc  (N.Z.),  B.Sc.  (Vict.). 
Crown  Svo,   7s.   &d. 

SPECTEOSCOPY.     By  E.  C.  C.  BALY,  F.I.C,  Lecturer  on  Spectroscopy 

and  Assistant  Professor  of  Chemistry,  University  College,  London.  With 
163  Illustrations.     Crown  Svo,  10s.  6d. 

The  following  Volumes  are  also  in  'preparation  : — 
EELATIONS   BETWEEN    CHEMICAL   CONSTITUTION   AND 

PHYSICAL  PEOPEETIES.     By  SAMUEL  SMILES,  D.Sc. 
STOICHIOMETEY.     By  SYDNEY  YOUNG,  D.Sc,  F.R.S. 
THEEMODYNAMICS.     By  F.  G.  DONNAN,  M.A.,  Ph.D. 
No.  6— 2,000— A.  u.  p.— ]/19(i7. 


DATE    DUE    SLIP 

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THIS  BOOK  IS   DUE   ON   THE   LAST   DATE 
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